The level with Eely-Mouth. After you clean his teeth, he spits out his golden tooth (actually a Shine Sprite) and leaves behind a heart made of coins. Eely-Mouth is saying thank you. Awwww.
When FLUDD seemingly passes away, Mario not only looks depressed but he also seems to be wiping away tears from his eyes. F.L.U.D.D may have simply been a robot with artificial intelligence to most people but to Mario he was a true friend.
The scene with Bowser and Bowser Jr. at the end, oddly enough. You can actually see a tear forming in Bowser's eye when he's trying to break it to Junior that Peach isn't his mother... only for his son to reveal that he knew the whole time that Peach wasn't really his mother And then we get this line.