Hundreds of police officers wearing riot gear used pepper spray to disperse the protesters, and arrested dozens of people.
Police said Lung Wo Road had to be cleared as it was a major thoroughfare.
After the scuffles, police said officers involved in beating a protester would be reassigned.
Demonstrators, a mix of students and a pro-democracy group called Occupy Central, have occupied parts of Hong Kong for more than two weeks. They are demanding fully free elections in the next vote for the territory’s leader.
China, which has control over Hong Kong, says residents can vote – but it will vet which candidates are eligible to stand.
The BBC’s Juliana Liu in Hong Kong says violent clashes have been a rare sight since the evening that tear gas was first used at the beginning of the pro-democracy protests.
It is the third day of operations that police say are necessary to ease traffic disruption, but which they insist are not aimed at clearing the protesters.
Their new advance came when protesters blockaded the underpass after being cleared out of other areas of the city on Tuesday.
Local television networks broadcast live footage of scuffles, showing police arresting many protesters, whose hands were tied with plastic cuffs.
Kicked and beaten
Tsui Wai-Hung, a police spokesman, said 37 men and eight women had been arrested for “unlawful assembly”.
None of those arrested had been hurt, the spokesman insisted, but four police officers were said to have been injured.
However, footage aired on local TV showed a protester who was handcuffed being kicked and beaten by police officers during the operation.
Secretary for Security Lai Tung-kwok said later on Wednesday that some of the officers seen on the video would be moved to other posts and that an investigation would be carried out.
The protesters are now in their third week of occupying key parts of the city in a bid to put pressure on China and Hong Kong’s authorities to answer their calls for reform.
Thousands of people took to the streets at the beginning of the demonstrations but the numbers have dwindled in recent days.
Over the weekend, Hong Kong’s embattled leader CY Leung said the protesters had zero chance of changing Beijing’s mind on constitutional reform, saying the protests had spun out of control.