Browns coach Kevin Stefanski said Walker will undergo an MRI.
"It's a huge loss with Anthony Walker if this is a longer-term thing," Brown coach Kevin Stefanski said. "We know what he brings to the football side of things, but he's a captain and he's a leader, so we got our fingers crossed and definitely hoping for the best for No. 5."
Walker suffered the injury during the third quarter. He laid facedown for several minutes before trainers put an air cast around his knee and helped him onto the cart.
The Browns also lost their other starting linebacker, Jeremiah Owusu-Koramoah, with a quad injury. Owusu-Koramoah spent several minutes in the medical tent before heading into the locker room.
Walker, 27, had six tackles against Pittsburgh before his injury. He had a team-leading 113 tackles and one sack in 13 games last season, his first with Cleveland.
The Browns won 29-17.