Padalecki announced the good news on Twitter, reported Aceshowbiz.
"We got a lot of questions this weekend about my wife and whether or not we wanted another child. Well, we want everybody to know that Genevieve is pregnant," he wrote.
The actor married his 'Supernatural' co-star in February 2010 in Sun Valley, Idaho. The couple welcomed their first child Thomas Colton 16 months ago.
"We've met a lot of new parents with little babies, and a lot of babies seem like babies, but he feels like he's kind of an old soul. He kind of looks at you and tries to figure you out, which is cool. I hope he's a mature kid," the actor had earlier said.