Jays manager John Gibbons indicated that there were no major injuries behind the moves.
Rios, who tweaked his back and groin in Monday's contest, was a gametime decision for Tuesday's game. He did play, however, and went 0-for-4 at the plate. His day off comes at a good time for the right fielder, as with the Jays not playing on Thursday, Rios would have two days in a row to rest.
Gibbons also said that Rolen and Barajas were just given regular off-days.
Rolen has been in a bit of a funk at the plate recently, recording only one hit in his last 14 at-bats. Barajas, on the other hand, has been swinging the bat well lately and has played in each of the Jays' last 14 games.
With the three hitters out of the lineup, Gibbons had the chance to spread some playing time around his bench. Curtis Thigpen replaced Barajas in the lineup and was also making his first start of the season for Toronto.
Marco Scutaro replaced Rolen at third and Kevin Mench started in left field, while Brad Wilkerson moved across from his regular position in left to fill in for Rios in right field.
David Singh is an associate reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.