Mountcastle was in the lineup batting fourth and playing first base at Tampa Bay. He had been out since he took a hard tag to the head while trying to steal second against the Rays on Aug. 6.
"Excited to have Ryan back," Orioles manager Brandon Hyde said. "He passed everything yesterday. He's ready to go."
Mountcastle was on an eight-game hitting streak and a seven-game RBI run when he was hurt. Baltimore hasn't won a game since then and has lost 12 straight overall.
Orioles first baseman Trey Mancini was out of Tuesday's lineup with a sore left calf. He fouled balls off his left calf and knee during an eighth-inning at-bat in Monday night's 9-2 loss to the Rays.
Hyde didn't rule out Mancini starting on Wednesday night.
"The calf was really sore when he woke up this morning," Hyde said. "He had treatment on it but it's still really sore."
Outfielder Ryan McKenna was optioned to Triple-A Norfolk.