Holland's Bauke Mollema won the 117-mile leg from Calahorra to Burgos after Tuesday's rest day in a time of four hours 44 minutes, breaking away from the group less than 500 metres from the end to edge out Team Sky's Norwegian rider Edvald Boasson Hagen.
But the Italian race leader was not far behind and prevented main rival Chris Horner, from the United States, gaining any more ground after losing 22 seconds on Monday.
Spanish duo Alejandro Valverde and Joaquin Rodriguez are third and fourth, over a minute and two minutes behind respectively, with Ireland's Nicolas Roche up to fifth, albeit 3:43 off the lead.
On Thursday the riders tackle a gruelling 116-mile mountain stage from Burgos to Pena Cabarga, with the race concluding in Madrid on Sunday.
"All day I was thinking about an attack in the last 1km because I knew, with the climb 8km from the finish, that the peloton was not going to be really big," Mollema told reporters.
"In the last 1km I was suffering so much, but I was just thinking that this is my chance and I have to take it."