Tied with overnight leader Igor Anton since Saturday, the Katusha rider snatched a two-second bonus early on the 175.7-km Stage 10 which was won by another fellow Spaniard, Imanol Erviti.
The bonus put Rodriguez ahead of Anton by two seconds, with Italian Vincenzo Nibali four seconds off the pace.
"Taking the jersey in itself is important but it's the long-term game I'm really interested in," Rodriguez told reporters. "I'm the sort of rider who's got to take whatever seconds are on offer wherever I can.
"I'm a lousy time trialist and there's a big time trial in the final week of the race. If there's a bonus on offer I'll fight for it."
Rodriguez was denied the race lead three days ago because Anton had recorded better results in previous stages.
"It's great to take the leader's jersey here in Catalonia, where I was born," said Rodriguez.
"But it's more important to get the jersey after being so close to it for so long and working so hard for it."
Erviti clinched the Caisse d'Epargne team's second straight win in the race after forming part of an early 11-man break which broke up on the lung-burstingly tough slopes of the Rat Penat climb.
He then sped away alone on the rough, twisting roads of the descent to the Catalan coastal resort of Vilanova i la Geltru.
Second was Romain Zingle, 37 seconds back, with his fellow Belgian Greg Van Avermaet in third spot.
"When a team here in the Tour of Spain has five leaders, like ours, it' harder to succeed than if there's only one or two," said Erviti.
"We take turns to keep an eye on the breaks and after (team mate) David (Lopez) won the other day, today it was my go. I could see the other guys had a lot of problems on that descent so I went for it."
Pre-race favorite Andy Schleck of Luxembourg failed to start the stage after Saxo Bank withdrew him from the race for breaking team rules.
Schleck said he had taken an alcoholic drink after supper with team mate Stuart O'Grady, who was also expelled.
The race finishes in Madrid on Sept. 19.
Meanwhile, two-time Tour de France runner-up Andy Schleck has been withdrawn from the Tour of Spain for breaking a team rule, the Luxembourg rider said on Tuesday.
A pre-race favorite, the Saxo Bank rider was lying 77th overall, more than 37 minutes down, before Tuesday's 10th stage. His Australian team mate Stuart O'Grady was also sent home by team manager Bjarne Riis.
"I acknowledge that I have broken a rule of the team by going out for a drink after dinner and for that reason Bjarne has decided to send myself and Stuart O'Grady home," Schleck was quoted as saying by Cycling News.
"I'm responsible for my actions and even though I think it is too harsh a decision, I respect that Bjarne is the boss and he needs to do what he thinks is the best."
Schleck's brother Frank remains in the race, lying 13th overall.
The Tour of Spain, which finishes on Sept. 19 in Madrid, is led by Spain's Igor Anton.
