Guaidó is currently President of the National Assembly, an elected legislature whose acts were declared null and void by Venezuela’s Supreme Court on Monday.
The National Assembly had refused to recognize the authority of President Nicolas Maduro, declaring his election victory last year illegitimate.
Guaidó took the oath of office in front of a crowd of supporters in Caracas on Wednesday, as thousands of Venezuelans thronged the city’s streets to protest Maduro.
He urged the country’s armed forces to support new elections, and pledged to perform the duties of president in the interim.
The call was rebuffed by defence minister, Vladimir Padrino who said the army will continue to defend the constitution and national sovereignty.
Moment after the news broke, US President Donald Trump said he recognizes Guaidó as Venezuela’s legitimate president, and encouraged other western powers to follow suit.
“The people of Venezuela have courageously spoken out against Maduro and his regime and demanded freedom and the rule of law,” Trump said in a statement.
“We continue to hold the illegitimate Maduro regime directly responsible for any threats it may pose to the safety of the Venezuelan people.”
Ahead of Wednesday’s protests, US Vice President Mike Pence released a video message reiterating the US’ commitment to regime change in Venezuela.
Denouncing Maduro as “a dictator with no legitimate claim to power” who has “never won the presidency in a free and fair election,” Pence vowed that the US “will not stand by as Venezuela crumbles.”
Maduro responded by breaking off diplomatic ties with the “imperialist government” of the US and giving the diplomatic mission 72 hours to leave the country.
“Get out! Leave Venezuela, here there’s dignity, damn it,” shouted Maduro to thousands of cheering supporters outside the presidential palace in Caracas.
A number of other countries were quick to follow Trump’s lead, with Brazil, Argentina, Colombia and Canada among those backing Guaido, although Mexico said it still considered Maduro the president.
Chile, Peru and Paraguay joined the ranks supporting Guaido while the head of the Organization of American States (OAS), Luis Almagro, congratulated the National Assembly president in a tweet.
“You have all our recognition to launch the return of democracy to the country,” he wrote.
The European Union merely said it was “following the developments in Venezuela very closely.”
A senior US administration official told journalists the country wouldn’t take action if the Venezuela regime used force against the opposition.
“If Maduro and his cronies choose to respond with violence, if they choose to harm any of the National Assembly members… all options are on the table for the United States in regards to action to be taken,” said the official.