Police cleared 30 to 40 people from inside Columbia University's Hamilton Hall on Tuesday, the Associated Press reported.
The protesters who took over the campus building on Tuesday barricaded and locked doors at the entrance.
Dozens of people were arrested by police. Those who were arrested were zip-tied and escorted to buses.
Columbia says decision to reach out to NYPD 'in response' to actions of protesters
"The decision to reach out to the NYPD was in response to the actions of the protesters, not the cause they are championing," a spokesperson for the university said in a statement.
"After the University learned overnight that Hamilton Hall had been occupied, vandalized, and blockaded, we were left with no choice," the statement read.
"We believe that the group that broke into and occupied the building is led by individuals who are not affiliated with the University," it added.
NYPD enter Columbia University
Special police units from the New York Police Department (NYPD) used a massive truck and a ramp to enter a building occupied by pro-Palestinian protesters at Columbia University in New York City on Tuesday night local time.
Officers took dozens of protesters into custody, with pictures showing police, wearing helmets and carrying zip ties and riot shields, massed at the Ivy League university's entrance in Manhattan.
Some protesters had made their way from an encampment to Hamilton Hall, the main academic building for undergraduates, and occupied the building for 12 hours on Tuesday.
Protesters were asked to leave their encampment by 2 p.m. local time on Monday afternoon. Some of those who refused had already been suspended.
"We made it very clear yesterday that the work of the University cannot be endlessly interrupted by protesters who violate the rules," Vice President of Public Affairs Ben Chang said in a statement on Monday.
Columbia has been at the epicenter of pro-Palestinian protests and students voted to defy the order to clear their encampment and stay.
Shortly before officers entered the campus, the New York Police Department received a notice from Columbia authorizing officers to take action.
Police cleared an encampment at the university on April 18 and arrested more than 100 people.