Defence Minister Gustavo Rangel said the jet had been tracked by country’s air defences over the Venezuelan-owned island of La Orchila on Saturday.
He said the US ambassador would be summoned to provide an explanation.
Venezuelan President Hugo Chavez, a fierce critic of Washington, has in the past accused the US administration of destabilising his country.
Mr Rangel told a news conference that the US aircraft “practically flew over” La Orchila – where Venezuela has a military base – and another island before turning back.
“We ordered the airplane to identify itself,” he added.
“We have recorded proof of the conversation between ground control in Venezuela and the aircraft pilot.”
The pilot is reported to have said he was on a training exercise and was unaware that he had entered Venezuelan airspace.
Asked about the allegation, US embassy spokeswoman Robin Holzhauer told the Associated Press news agency: “We’re looking into any possible accidental incursion of Venezuelan airspace.”
Mr Rangel said the incident was “the latest step in a series of provocations” by the US.
The BBC’s James Ingham in Caracas says it comes at a time of increased tension in the region, with both the US and Colombia accusing Venezuela of financing the left-wing Revolutionary Armed Forces of Colombia (Farc).
Last week Interpol confirmed that computer evidence found by Colombian soldiers during a raid on a Farc camp showed no sign of tampering.
Colombia says various documents proved that Venezuela has funded the rebels. Mr Chavez insists he never provided any support.
Venezuela’s accusations against the US come two days after a similar claim was made against Colombia.
The Bogota government denied that its troops had crossed into Venezuelan territory on Friday.
Our correspondent says the latest row will further increase tension between Venezuela and two countries which it clearly sees as enemies.