In 1938, 49 years after the events of Phantom Blood, Robert E.O. Speedwagon, who got rich and founded the Speedwagon Foundation that dedicates itself to medical and enviromental research, and Straizo, who succeeded Tonpetty as a master of Hamon, travel to Mexico to explore a mysterious cavern, filled with many stone masks similar to the one that turned Dio Brando into a vampire, and a body of a man integrated into a stone pillar. Straizo unexpectedly betrays Speedwagon and their companions, kills them and injures the former. Envy of Dio's immortality and youth, Straizo dons a stone mask to becomes a vampire and be able to look young and live forever.
Meanwhile, Jonathan Joestar's widow and grandson, Erina and Joseph Joestar, are in New York. Joseph's wallet is stolen by a thief, who's beaten by two policemen for that. Joseph saves the young thief, named Smokey, using Hamon. He introduces himself to him and explains that he inherited this ability from his grandfather. Later, Joseph, Erina and Smokey all go to a restaurant. After Joseph fights a racist Mafioso, showing off his skills of predicting his opponents, the Mafioso's acquaintance informs Joseph that Speedwagon has been murdered by Straizo. Later that night, Joseph is confronted by the now-vampire Straizo, and blasts him with a Tommy Gun. They fight, and after Joseph defeats Straizo, the latter tells him about the Pillar Man that is about to be awakened before self-destructing himself with Hamon.
Speedwagon has been found by Nazi soldiers and taken to their base in Mexico. An officer named Rudol Von Stroheim interrogates Speedwagon about the Pillar Man, revealing to him that the Nazis have already captured him. The Nazis sacrifice a group of Mexican prisoners to awaken the Pillar Man. Meanwhile, Joseph makes his way to mexico, where he's ambushed by a Nazi assassin named Donovan. Joseph defeats Donovan, who tells him that Speedwagon is still alive and is held at the Nazis' base. Joseph arrives at the base and after a failed attempt of disguising himself as a woman to get in, steals the uniform of a soldier and sneaks in. In the base, the Nazis have succeeded awakening the Pillar Man, and Stroheim names him Santana. Stroheim sends a vampire made by the Nazis to challenge Santana, and the Pillar Man absorbs him into his own body. Santana escapes the cell he was put in and enters the body of one of the soldiers. The disguised Joseph arrives at the lab, relieved to see Speedwagon alive, and reveals himself before he starts fighting Santana.
Joseph tries to makes peace with the Pillar Man in a playful way, but changes his mind after Santana tries to attack Speedwagon. Joseph manages to cut Santana in half using Hamon, ties him to a chain and drags him to the base's entrance, trying to expose him to the sunlight and destroy him. Stroheim tries to help by asking Joseph to cut his leg. Stroheim opens the entrance door, but Santana shields himself from the sunlight by entering Stroheim's body through his wound. Stroheim blows himself up to expose Santana to the sunlight again, but not before telling Joseph that three other Pillar Men were uncovered in Europe. He tells him to go to Rome, Italy, where he'll meet someone important. Santana's body is taken to the Speedwagon Foundation's headquarters, where they find out that the other three Pillar Men may be stronger than him.
Joseph and Speedwagon arrive in Rome, where they meet the man Stroheim talked about - Caesar Anthonio Zeppeli, grandson of Will Anthonio Zeppeli, Jonathan Joestar's ally and mentor. Joseph and Caesar don't get along well. All three wait in a hotel until a car arrivers, and a German soldier named Mark, with whom Caesar allied so the Germans can study Hamon in order to find a way to defeat the Pillar Men, takes them to a colloseum where the other three Pillar Men were found. Before they arrive, the three Pillar Men - Wamuu, Esidisi and Kars - have already awakened, and killed all the German soldiers in the area.
Joseph's group arrives at the area, and they find out that the Pillar Men have awakened. Soon enough, they encounter all three of them. Wamuu walks into Mark, killing him and enraging Caesar. The Pillar Men explain that they are looking for the Red Stone of Aja, which they can insert into a stone mask and overcome their weakness to the sun. Caesar tries to fight Wamuu with his bubble Hamon, but Wamuu injures him. Joseph fights the Pillar Man, luring him out so they won't harm the others. Wamuu overcomes him, but Joseph claims that he'll be able to rematch him after 33 days of Hamon training. Wamuu implants a poisonous ring inside his body that will disintegrate in 33 days, releasing poison that can only be cured with an antidote stored inside his lip piercing. Esidisi implants another poisonous ring inside Joseph's body, with the antidote stored in his own nose piercing. Caesar sees what happened and promises to train with Joseph.
Joseph and Caesar go to Venice, where they meet their new Hamon teacher, a woman named Lisa Lisa. They arrive at Air Supplena island, where Lisa Lisa tasks them to climb the Hell Climb Pillar, a 24-meter pillar covered in oil, using only Hamon. After days of climbing, Joseph notices a crack in the pillar which he can use to climb, but it turns out to be a trap, causing the pillar to rapidly spew oil from it's 20 meter mark. Caesar manages to get to the top of the pillar, but worries that Joseph might not be successful. With the help of Caesar, Joseph manages to climb the pillar. Lisa Lisa reveals to the boys that she possesses a Red Stone of Aja, and she and her assistants, Loggins and Messina, begin to train the boys in Hamon.
After almost a month of training, Lisa Lisa pits Joseph and Caesar against Loggins and Messina respectively. While Caesar duels Messina, Joseph arrives at the arena where he's supposed to fight Loggins, only to find out that he has been killed by Esidisi. Joseph defeats the Pillar Man, showing how he progressed in his Hamon training, and takes the antidote from his nose piercing. Joseph walks back to the training guard, unware of Esidisi's brain clinging to his back, and meets Lisa Lisa's assistant, Suzi Q. Without Joseph noticing, Esidisi's brain enters Suzi's body, possessing it and using it to deliver Lisa Lisa's Stone of Aja on a boat. Joseph, Lisa Lisa and Caesar try to find a way to defeat Esidisi without killing Suzi Q. Joseph and Caesar use their combined Hamon to force Esidisi's brain out of Suzi's body, releasing it and letting the sun disintegrates it. Suzi Q reveals that the stone is aboard a train headed to Switzerland, so Joseph, Caesar, Lisa Lisa and Messina go to retrive it.
The groups arrives at the train and they find out that the Nazis have come to retrieve the stone for research. A Nazi soldier tells the group to come with him to a log cabin up ahead. Kars arrives at the cabin and kills the soldiers in it, short for one. Kars wonders how he couldn't sense the soldier's body heat, and the soldier reveals himself, to Joseph's shock, to be a now-cyborg Stroheim. Stroheim fights Kars, who takes the Stone of Aja but then drops it, causing it to slide towards a cliff. Joseph and Kars race for the stone. As Kars can survive the fall from the cliff, he takes the stone, falls from the cliff and takes Joseph with him, but Joseph manages to get back over, taking the stone with him. A day later, the group finds the hotel where Kars and Wamuu reside. Caesar suggest to attack the pillar men in the morning, but Joseph suggest it might be wiser to do so at night. The two fight about the matter, and Caesar decides to fight Kars alone, disobeying Joseph and Lisa Lisa's request, with Messina accompanying him.
Lisa Lisa tells Joseph of Caesar's past: In his childhood, his father, Mario, has left him and his siblings. Angry at his father, Caesar became a criminal, committing many crimes expect for murder. When Caesar turned sixteen, he saw his father observing the wall where the Pillar Men were contained. A trap within the wall is activated, and Mario sacrifices himself to save Caesar, and tells him to meet Lisa Lisa and learn to use his Hamon. Joseph decides to head to the hotel with Lisa Lisa. At the hotel, Wamuu takes Messina down and fights Caesar. Caesar manages to divert sunlight into the hotel using his bubbles Hamon, but Wamuu attacks him with a Divine Sandstorm, gravely injuring him. Caesar steals Wamuu's mouth piercing and ties it to his headband. In his last breath, Caesar continues the Zeppeli family's legacy and creates a blood bubble so he can deliver the ring to Joseph, before a large rock crushes his body.
Joseph and Lisa Lisa get in the hotel and find the bubble and Caesar's blood flowing from under the rock. After grieving their fallen friend, they continue to explore the hotel, and Joseph decided not to take the antidote inside Wamuu's piercing until he defeats the Pillar Man. Joseph and Lisa Lisa find Wamuu, Kars, and 100 other vampires in a dark room. Lisa Lisa lies that she rigged the Red Stone of Aja with a bomb, and that killing her will cause it to explode. The Hamon users and the Pillar Men decide to meet in an arena at midnight and fight there one on one - Joseph vs. Wamuu, and Lisa Lisa vs. Kars - for the stone. Joseph goes back to the hotel to retrieve the stone, and finds a picture album in Lisa Lisa's luggage, with a picture of Erina, Speedwagon, Straizo and a baby in it. When Joseph returns to the Arena, Lisa Lisa reveals to him that she's the baby in the picture, and that she was raised by Straizo.
Joseph and Wamuu have a chariot race, and they fight each other. Joseph defeats Wamuu, taking the antidote and saluting for the honorable Pillar Man. Kars takes Joseph and Lisa Lisa to a temple, where he'll fight the latter. However, Lisa Lisa notices that Kars doesn't intend to kill her, and after attacking him, another Kars comes from behind her and stabs her, revealing the one she fought to be a clone. Kars takes the Stone of Aja and orders his vampires to attack Joseph. He also hangs Lisa Lisa above a field of crystal spikes, but Joseph is rescued by Speedwagon, Stroheim, Smokey and members of the German Army and the Speedwagon Foundation, who fight the vampire army using UV lights. Joseph manages to throw Kars into the field of spikes and saves Lisa Lisa.
Meanwhile, Speedwagon tells Smokey that Lisa Lisa is Joseph's mother, whose real name is Elizabeth. He tells him about how she married Erina's son, George Joestar II, a Royal Flying Corps member. He tells Smokey how Lisa Lisa avenged his death by the hands of a zombie that disguised himself as an RFC officer and killed George. Lisa Lisa was labeled a murderer because the other RFC members didn't knew their commander was a zombie, so she left Joseph at the care of Erina, who asks Speedwagon to never tell Joseph the truth about his parents.
Meanwhile, Stroheim attempts to finish Kars off with the UV lights, but Kars reveals that he has already placed the stone of Aja inside a stone mask and donned it. Kars becomes the ultimate being: He can transform part of his body into different living things, and the sunlight can no longer destroy him. Joseph grabs the Stone of Aja and runs away with Smokey, with Kars growing bird wings to chase them. Joseph boards a German war plane and lures Kars into a volcano. Kars crashes the plane, and Joseph and Stroheim, who was hiding inside the plane all that time, land on the ground, with Kars falling inside the volcano. Kars survives the hot lava, severs Joseph's left arm and injures his leg using Hamon. Joseph holds the Stone of Aja in front of him as Kars attacks him, causing the volcano to erupt, and they're both launched into space. Kars is launched further from the earth's atmosphere and his body freezes, while Joseph crashes back into the sea. On February 28, 1939, Joseph Joestar is pronounced dead.
Two months later, back in New York, a funeral is held for Joseph, attended by Erina, Smokey, Speedwagon, Lisa Lisa and Messina. Joseph, now fitted with a new prosthetic arm created by Stroheim, crashes the funeral, leaving everyone shocked that he's still alive. Suzi Q arrives as well, with a wedding ring on her finger. Jojo tells his friends that he and Suzi Q are married. Suzi Q reveals that she forgot to telegraph them about both matters.
For the several next years, all of them continued with their lives. Lisa Lisa tells Joseph that she's his mother, and together with Suzi Q they move to New York, where Lisa Lisa married a screenwriter. Erina continued her work as a teacher until her death in 1950, Speedwagon continued to manage the Speedwagon Foundation until his death in 1952, Smokey studies Political Science in college and becomes the first black mayor of his hometown in Georgia, and Stroheim died in 1943 in the battle of Stalingrad.
In 1987, Joseph, now an old man, is at the airport, waiting to board a flight to Japan to visit his daughter. He developed a grudge against Japanese people after his daughter married a Japanese man and moved far away from him. He boards the flight to visit her and his grandson.
