
Reports say hundreds of police officers wearing riot gear used pepper spray to disperse the protesters, arresting dozens of people in the process.
At a briefing before the clashes, police said they had to clear Lung Wo Road as it was a major thoroughfare.
Demonstrators have occupied parts of Hong Kong for more than two weeks.
The protesters, a mix of students and a pro-democracy group called Occupy Central, 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.
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 hours after 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.
'Punches, kicks, elbows'
Tsui Wai-Hung, a police spokesman, said 37 men and eight women had been arrested for "illegal assembly".
None of those arrested had been hurt, the spokesman insisted, but four police officers were said to have been injured.
However, footage shown on local station TVB appeared to show one protester being taken aside by police officers and beaten.
Daniel Cheng, a journalist reporting on the clashes, told the AFP news agency that he was also beaten up by police officers.
"[Police] grabbed me, more than 10 police, and they beat me: punches, kicks, elbows. I tried to tell them I'm a reporter but they didn't listen."
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.
BBC/TF