Nicolas Maduro accused the three unidentified U.S. consular officials of infiltrating Venezuelan universities under the guise of working on student visas.
But instead of doing consular work, they fomented a growing upsurge in student protests that have resulted in three deaths and scores of injuries, Maduro charged in a televised address Sunday night.
"It's a group of U.S. functionaries who are in the universities. We've been watching them having meetings in the private universities for two months. They work in visas," he said.
He said the Foreign Ministry would provide details later.
"Venezuela doesn't take orders from anyone!" the BBC quoted him as adding.
The State Department had no immediate comment.
Maduro's address came after Secretary of State John Kerry expressed concerns about Venezuela's escalating tensions.
"We are particularly alarmed by reports that the Venezuelan government has arrested or detained scores of anti-government protesters and issued an arrest warrant for opposition leader Leopoldo Lopez," Kerry said in a statement Saturday.
"We urge all parties to work to restore calm and refrain from violence," he said.
The regime blames "right-wing fascists" for the protests and has a manhunt out for Lopez, who it accuses of inciting the bloodshed.
Student organizer Gaby Arellano said blame for the growing violence laid plainly in the hands of pro-regime vigilante groups known as "colectivos," which Arellano accused of acting with impunity.
"They're the ones responsible for the chaos and anarchy not only in Caracas but all of the national territory," she told demonstrators Sunday in remarks quoted by the Miami Herald. "They're the ones who have made families mourn in the last days and now the [regime] wants to blame Leopoldo Lopez for the situation."
The Caracas daily Ultimas Noticias also questioned the official version of the most serious riots Wednesday.
Lopez posted a video on YouTube Sunday in which he denied being behind the violence and asked followers to join him in a peaceful protest Tuesday as he marches to the state prosecutor's office in downtown Caracas, the capital, to demand the regime release students jailed in demonstrations two weeks ago and stop persecuting protesters in general.
Lopez, 42, a Harvard-educated former mayor of a Caracas district who the late President Hugo Chavez banned from running for public office until this year, said authorities could apprehend him during Tuesday's march they wanted.
"I have nothing to fear. I've done nothing wrong," Lopez said in the 3-minute-plus video.
"If there is any illegal decision to jail me, then I will accept that decision and that infamous persecution by the state," he said.
He asked for demonstrators to "go dressed in white to signify our commitment to peace."
Shortly before Lopez's video was posted, Henrique Capriles, an opposition leader who lost to Chavez in the 2012 presidential elections and then to Maduro last year, said he would call for a national march against criminal gangs and violence this week.
Capriles did not set a date for his march.
Maduro Sunday repeated his assertions the opposition was behind the violence.
"Venezuela has the right to defend itself from fascist gangs," he said. "We will defeat those gangs."
He suggested those protesting could possibly lead to a coup -- to which Capriles said: "Civilians do not stage coups, so we demand the government to show evidence of those in the military who may be acting against the Constitution."