Milledge said he injured himself when he got back up to get the ball as it rolled toward the wall.
"If you look at the tape, I got caught picking it up," Milledge said. "I got caught, I dropped the ball, and then picked it up and threw it, and it was pulled."
Manager Manny Acta and assistant athletic trainer Michael McGowan ran out to take a look at Milledge, and it became clear he could not continue. Willie Harris came in to play center field in Milledge's place.
The Nationals called up outfielder Roger Bernadina from Double-A Harrisburg to take Milledge's place on the roster.
After the game, Milledge didn't hide his disappointment about picking up the injury.
"I played pretty much in every game except for one game this year," a somber Milledge said. "I had planned on playing at least 160 games this year."
Acta indicated that Bernadina would start in center field on Sunday, and that he would get a chance to be the everyday center fielder in Milledge's absence.
"We're going to give him a shot to play center field," Acta said.
Still, Acta said he knows losing Milledge will hurt, especially considering the young center fielder's strong play of late.
"The kid was leading our club in RBIs, and he was making so much progress in center field, he was looking so good and looking comfortable hitting third," Acta said, adding that Milledge is expected to be out two to four weeks. "It's been that kind of year, we'll give another kid a chance."
Coming into action on Saturday, the 24-year-old Bernadina was hitting .325 with five home runs and 38 RBIs at Harrisburg. In addition to hitting 11 doubles and seven triples, Bernadina also displayed speed on the basepaths, stealing 26 bases. His speed and defensive play led Baseball America to rate him Washington's best defensive outfielder and fastest baserunner in the Minor Leagues.
Bernadina has been particularly effective leading off an inning. In those situations, Bernadina is hitting .404 with four home runs, six walks and a .440 on-base percentage.
When roving instructor Devon White was in Washington earlier during the current homestand, he singled out Bernadina as a Nationals Minor Leaguer he has been impressed with this year.
"I'm very high on Bernadina," White said. "He's very fast. ... He's still learning the game, he's been playing six years."
Milledge's injury means that Christian Guzman is the only Opening Day starter not to spend time on the DL.
"It's tough for the team," Milledge said. "It's another starter that goes down. It's kind of tough right now, the situation that we're in."
Milledge was honest when asked how he felt about going on the disabled list.
"I grew up playing every game, and that's how my dad raised me," Milledge said. "I'm pretty sure if he was here right now, he'd probably throw me out there tomorrow if I was in Little League. But it's a business and we're in the big leagues, and we've got to kind of take things one day at a time."
Zachary Osterman is an associate reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.