The US has warned Venezuela that any threats against American diplomats or opposition leader Juan Guaidó will be met with "a significant response".
National Security Adviser John Bolton said any such "intimidation" would be "a grave assault on the rule of law".
His warning comes days after the US and more than 20 other countries recognised Mr Guaidó as interim president.
Meanwhile, Mr Guaidó has called for anti-government protests on Wednesday and Saturday.
Mr Guaidó, the elected leader of the opposition-held National Assembly, declared himself the interim president on 23 January.
The political crisis in Venezuela now appears to be reaching boiling point amid growing efforts by the opposition to unseat President Nicolás Maduro.
He was sworn in for a second term earlier this month after an election marred by an opposition boycott and allegations of vote-rigging, triggering large protests.
On Sunday, Venezuela's top military representative to the US, Col José Luis Silva, defected from Mr Maduro's government, saying he recognised Mr Guaidó as president instead.
Later, Mr Bolton took to Twitter to reiterate Washington's position, warning others against any form of "violence and intimidation".
Cuba’s support and control over Maduro’s security and paramilitary forces are well known. 1/2— John Bolton (@AmbJohnBolton) January 27, 2019
Any violence and intimidation against U.S. diplomatic personnel, Venezuela’s democratic leader, Juan Guiado, or the National Assembly itself would represent a grave assault on the rule of law and will be met with a significant response. 2/2— John Bolton (@AmbJohnBolton) January 27, 2019
Also on Twitter, Mr Guaidó called for a "peaceful" two-hour strike on Wednesday and a "big national and international rally" on Saturday.
What happens now?
On Saturday, several European countries including Spain, Germany, France and the UK said they would recognise Mr Guaidó as president if elections were not called within eight days.
But Mr Maduro has rejected this, saying the ultimatum must be withdrawn.
"Venezuela is not tied to Europe. This is complete insolence," he told CNN Turk on Sunday.
Mr Maduro added that he was ready to "engage in comprehensive dialogue" with those who opposed his presidency. He said he had sent Donald Trump "many messages", but he thought the US president "despises us".
He later appeared at a military exercise in Venezuela's central state of Carabobo, where he called for "union, discipline and cohesion" to overcome what he described as an "attempted coup d'etat" by Mr Guaidó.
Mr Maduro broke off relations with the US last Thursday over the country's support for Mr Guaidó, and ordered US envoys to depart Venezuela within 72 hours.
However on Saturday evening, as the deadline was due to expire, Venezuela's foreign ministry said it would withdraw the expulsion order, and instead allow 30 days for the two sides to set up "interest offices" in each others' countries.
Interest offices are used when countries do not have formal diplomatic relations, but want to have a basic level of contact to represent their interests.
Washington has previously said it does not recognise Mr Maduro's authority to order its diplomats out.
Meanwhile, Mr Guaidó told the Washington Post that he was in talks with "sympathetic military" officials in Venezuela with the aim of building support for his presidency.
Venezuela's army has stood by Mr Maduro throughout previous upheaval, encouraged by regular wage increases and other incentives. In July, some 16,900 soldiers were promoted as a reward for their "loyalty".
Despite that, reports suggest the lower ranks are increasingly dissatisfied with the government, which they blame for food and medicine shortages, as well as frequent power and water cuts.
Who supports Maduro?
Russia, China, Mexico and Turkey have publicly backed Mr Maduro.
At a UN Security Council meeting on Saturday, Russia accused Washington of plotting a coup.
However, more than a dozen Latin American countries and Canada have backed Mr Guaidó as president.
In Europe, Greece's left-wing governing party has backed Mr Maduro.
Why is Maduro so unpopular?
Venezuela is in economic crisis - hyperinflation and shortages of basic essentials have hit its population hard and caused millions to flee.
Mr Maduro has faced internal opposition and ongoing international criticism for his human rights record and handling of the economy.
He was re-elected to a second term last year - but the elections were controversial, with many opposition candidates barred from running or jailed.
Supreme Court judge Christian Zerpa fled to the US in January, telling journalists the election "was not free and competitive".