Nicolas Maduro‘s regime blocked the Tienditas International Bridge with a giant orange tanker, two large blue containers and makeshift fencing.
Armed soldiers stood guard at the customs building, with instructions to turn back any attempt to cross the border.
Despite widespread hunger and a shortage of medicine, President Maduro vowed to turn back US aid after Trump’s administration recognised opposition leader Juan Guaido as the South American country’s rightful interim leader.
Mr Maduro argues Venezuela is not a nation of “beggars” and has rejected the idea of receiving humanitarian assistance, regarding it as a foreign intervention.
Mr Guaido said the emergency shipment was a “test” for Venezuela’s armed forces, which will have to choose if they will obey their orders or allow the much needed aid to pass.
International pressure is growing on Mr Maduro to step down after the UK and its major EU allies joined the US, Canada and a group of Latin American countries in recognising Mr Guaido as Venezuela’s legitimate leader following Mr Maduro’s re-election last year in a vote critics have called a sham.
Soaring hyperinflation has forced millions of Venezuelans to flee or go hungry as they struggle to find or afford basic goods.
US secretary of state Mike Pompeo said Venezuelans desperately need the emergency supplies the US and other countries are preparing to provide.
“Venezuela’s military under Maduro’s orders is blocking aid,” Mr Pompeo tweeted. “The Maduro regime must LET THE AID REACH THE STARVING PEOPLE.”
President Maduro has clung to power with the support of Venezuela’s highest-ranking military officers. He has dismissed Mr Guaido as a puppet of the US, which he says is seeking to colonise Venezuela and exploit its vast oil resources.
In his State of the Union address on Tuesday night, President Trump vowed to ratchet up the pressure on Mr Maduro, saying the US stands with the people of Venezuela.
“We condemn the brutality of the Maduro regime, whose socialist policies have turned that nation from being the wealthiest in South America into a state of abject poverty and despair,” Mr Trump said.
Additional reporting by agencies