Green Goblin- The Amazing Spider-Man #263

A little back story first: In 1995's The Amazing Spider-Man #400 Aunt May dies of health problems. There are a few touching moments leading up to her death, where May reveals that she has known about Peter being Spider-Man for a while. (If you're wondering, May finds out about Peter being Spider-Man in The Amazing Spider-Man Volume 2 #35) She says she is proud of him blah blah blah.

The Green Goblin becomes relevant to this story in 1998's The Amazing Spider-Man #263 when he reveals that May didn't die. Actually, she was kidnapped by Green Goblin and held for three years... Just to toy with Peter's emotions. After losing so many people in his life, it is incredible to consider the depth of suffering Green Goblin caused Peter by faking Aunt May's death.

Also worth mentioning: Aunt May actually does really die in The Amazing Spider-Man #543. She gets shot (in The Amazing Spider-Man #538) by one of Kingpin's assassins after Spider-Man reveals his secret identity, showing support for the Superhuman Registration Act, in the Civil War series.  She is saved, last minute, by Spider-Man's deal with the Devil (Mephisto) in Spider-Man: One More Day.