In what issue did Spiderman and Mary Jane break up?

Spider-Man and Mary Jane's relationship has been a long and complicated one, with multiple breakups and reconciliations throughout the comics. It's difficult to pin down one specific issue as the definitive "breakup" because their relationship has gone through several major shifts.

However, one of the most significant and lasting breakups occurred during the "One More Day" storyline, which ran from Amazing Spider-Man #544-546 in 2007. In this arc, Spider-Man makes a deal with the devil (in this case, Mephisto) to save Aunt May's life. As a consequence, Mephisto erases the marriage of Peter Parker and Mary Jane Watson from existence.

This breakup had a profound impact on Spider-Man's life and his relationship with Mary Jane, and it took years for them to reconnect and eventually rekindle their relationship.

Therefore, while there have been other significant breakups throughout their history, the "One More Day" storyline is often considered the most pivotal and lasting breakup between Spider-Man and Mary Jane.