Longoria ran into a 2-1 pitch from Blue Jays right-hander Esmil Rogers and deposited the ball into the left-field stands to pull him even with Pena.
"It's a proud moment for me, because this has been my home for so long and obviously I've made a significant commitment," Longoria said. "And obviously, knowing that I have a love for this franchise and this community, to be able to do it, you know, it's just one home run in the grand scheme of things. For what it means for this organization and for what it means to me, it's big."
Longoria's home run also broke open their 7-2 win over the Blue Jays.
"That's obviously the more important thing," Longoria said. "At that point of the game, we're really trying to tack a couple of more runs on and give a little breathing room to the bullpen. So I was able to afford that opportunity tonight."
Longoria's first career home run came against Brian Bruney of the Yankees on April 14, 2008, during an 8-7 loss at Tropicana Field.
Pena's 163rd -- and final -- home run with the Rays came against Boston's Jon Lester on Sept. 26, 2012, during a 4-2 Rays win.
Bill Chastain is a reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.