Horner, who is already cycling's oldest Grand Tour stage winner and race leader after his stage three triumph at Lobeira last weekend, proved equally unstoppable on Monday's 10th stage.
The RadioShack veteran attacked four kms from the summit of the Alto de Hazallanas ascent to stop the clock at the summit of the sun-drenched 15 kms climb, 48 seconds ahead of race favourite Vincenzo Nibali.
Spain's top contender Alejandro Valverde was in third place
With the first rest day scheduled for Tuesday, Horner leads by 43 seconds from Nibali with Ireland's Nicolas Roche third 53 seconds further back.
"I knew if I attacked and got 10 or 15 seconds then they would start playing games and start marking each other," Horner, a rider with the RadioShack-Leopard squad who turned professional in 1995, told reporters.
"Nibali was the strongest there, but things got very tactical."
Horner was pessimistic about his chances of retaining the lead in Wednesday's 38.8 kms time trial in Tarazona.
"It's been a long time, a good couple of years, since I did a good time trial," he said.
"I expect Nibali will get the (leader's) jersey there. But sometimes, just sometimes, I do do a good time trial."
After making the pace on the lower slopes of the final climb, and seeing overnight leader Dani Moreno of Spain fall back, Nibali staged a lone, fruitless, pursuit of Horner.
Asked if he had wanted the lead, Nibali responded: "I always do, but Horner did a brilliant ride.
"I'm not too worried, though, we're not even halfway through yet and the hardest part of the Vuelta is yet to come."
Although none of their riders won and their best rider, Spain's Igor Anton, finished eighth on the stage, the Euskaltel-Euskadi team were all smiles at the finish.
They had been told that their 17-year-old team would no longer fold at the end of 2013 with Formula One driver Fernando Alonso, a long-standing fan of the sport, poised to buy the team's licence for the WorldTour, cycling's top league.
"This news is way better than any victory" said team leader and 2008 Olympic gold medallist Samuel Sanchez of Spain.
"Now, at least, we can get on with the Vuelta without being worried about our jobs for next year."
The race finishes in Madrid on Sept. 15.