The Sand Bird ride. You run around the Sand Bird, collecting Red Coins, while the Sand Bird wonderfully screeches like a hawk.
In Pianta Village, after getting all red coins, and when you're on the gigantic palm tree, you grab a piece of "fluff" (a giant dandelion seed), then it flies all the way to a cloud that has the Shine out in the middle of the sky.
Bowser Jr. managing to get Mario framed for polluting Isle Delfino; even though it was largely Bowser's idea, The Reveal of Shadow Mario's true identity really establishes how cunning Bowser Jr. can be when he puts his mind to stopping Mario.
The first appearance of Mecha-Bowser, combined with the Crowning Music of Awesome and the fact that he actually breathes fire at the screen