Winning the seat is a key requirement for Anwar to succeed 93-year-old Prime Minister Mahathir Mohamad, who jailed his former protege and heir apparent on sodomy and corruption charges in 1998 when their relationship soured.
Mahathir returned to the premiership this year after a shock election win, saying he would stay in power for only two years before handing the reins to Anwar.
Anwar was in prison when he forged an unlikely alliance with Mahathir in a bid to unseat then prime minister Najib Razak, who had called elections for May amid massive corruption allegations.
Underscoring the drama of Saturday's vote, one of Anwar's six challengers is a former aide who also accused the then-opposition leader of sodomy, landing the 71-year-old in jail for a second time in 2014.
But the charismatic politician is expected to eke out an easy win in the seat, which was vacated after a member of the ruling coalition stepped down to pave Anwar's return.
Polls opened under cloudy skies at 8:00 a.m. (0000 GMT) in the sleepy southern coastal town of Port Dickson, home to a sizeable ethnic Chinese community that across the country has traditionally been one of Anwar's pillars of support.
"We are voting for the next premier. We need an influential leader to bring long-overdue progress to Port Dickson," said 60-year-old voter Lee Tian Hock.
"This morning, I prayed to Allah for a big win for Anwar," retired truck driver Mat Taib, a member of the country's ethnic Malay majority, told AFP.
"I want him to be our eighth prime minister."
About 100 supporters greeted Anwar with shouts of "Reformasi" -- his battlecry while in opposition -- as he arrived at a polling station.
"Voter turnout is too slow, Hopefully, more will come out to cast their votes after they finish their work in hotels and factories," he said.
"I will see you in parliament on Monday," he told an AFP reporter, smiling broadly.
Charismatic politician
Anwar campaigned hard over the past two weeks to secure a mandate in the multi-racial constituency, promising voters development, clean government and a boost to local tourism.
He has not discussed the accusations of sodomy -- an act that is still illegal in largely Muslim Malaysia -- while on the campaign trail. He has always maintained the charges were trumped up to derail his political career.
But he has campaigned doggedly on the multi-billion-dollar graft scandal at state fund 1MDB, over which former leader Najib and his wife Rosmah Mansor facing dozens of corruption charges.
Both face the prospect of spending the rest of their lives in jail in a scandal that saw Najib's coalition lose office for the first time since the country declared independence from Britain in 1957.
Political heavyweights including Mahathir have campaigned for Anwar in a road back to office that was unthinkable even six months ago.
The duo went onstage together at one campaign event, prompting wild cheers from supporters.
After he was dumped as finance minister and jailed in the 1990s, Anwar led a reformist opposition movement while fighting to overturn his convictions.
Mahathir, his mentor turned tormentor and now ally, came back from retirement to lead the Alliance of Hope coalition that won power in May.