Goals from Jozy Altidore and Clint Dempsey ended Spain's world record run of 15 successive victories and their 35-match unbeaten sequence, a world record streak they share with Brazil, and caused one of the biggest soccer upsets in years.
However, the match ended in bitter-sweet agony for U.S. coach Bob Bradley when his son Michael, who had a brilliant game in midfield, was sent off three minutes from time.
He was the third U.S. player sent off in the tournament and now misses Sunday's final against Brazil or hosts South Africa.
It was also the first time the U.S. had beaten a team ranked number one in FIFA's world rankings since they defeated Brazil in 1998.
"That was a great team effort, to beat a team like Spain, it's big," coach Bradley told reporters.
"We played as hard as we could, everyone played their part. We will take a few hours to regroup now but we will be ready for the final. We are so excited about this."
Counterpart Vicente Del Bosque, who had guided his side to 13 wins in a row since taking over after Spain won Euro 2008, said: "We are not used to losing any longer.
"Every defeat hurts. Today we needed the final touch. We had many opportunities but they really closed the door on us."
Altidore, 19, said: "The fact I play my football in Spain at Villarreal was a big help. I have played against a lot of these guys and know them and gave the coach some information.
"I sent some of them texts before the game telling them we were going to win. I was right. But everything went for us tonight, it was our night."
