"If those in power think that they can evade the benefits of real reform by stalling and procrastination, they are delusional," Sistani said in his weekly sermon, delivered by a representative in the Shia holy city of Karbala.
"What comes after these protests will not be the same as before, and they should be aware of that."
Since starting on October 1, demonstrations have escalated into demands for root-and-branch reform of the political system.
Sistani cautiously backed the protests when they began but has since firmed up his support, describing protests on Friday as "the honourable way" to seek change.
The 89-year-old cleric, who is based in the Shia holy city of Najaf and never appears in public, remains hugely influential in the Shia-majority south.
Emboldened after his sermon, thousands of protesters rallied in the southern hotspots of Kut, Hilla, Nasiriyah and Basra, AFP correspondents reported.
Near the capital's main protest camp in Tahrir (Liberation) Square, demonstrators decided to hold their ground after hearing the Shia religious leadership, or "marjaiyah".
"No one retreat, even the marjaiyah is with us!" said one young man as security forces pelted them with tear gas canisters.
In neighbouring Khallani Square, two protesters were shot dead on Friday afternoon, according to a medical source, after one was killed overnight.
A third protester was killed later in the day in the square by a tear gas cannister, the same source said.
More than 330 people have died since the rallies erupted, making them the deadliest grassroots movement to hit Iraq in years.
They present the biggest threat so far to the political system ushered in by the US-led invasion which toppled the regime of longtime dictator Saddam Hussein in 2003.
Protesters blame that system for rampant corruption, staggering unemployment rates and poor services in resource-rich Iraq, OPEC's second-biggest producer.
But the political establishment has rejected demands for the government to step down and instead closed ranks.
That consensus was brokered by neighbouring Iran's pointman for Iraq, senior Islamic Revolutionary Guard Corps commander Major General Qasem Soleimani. Sistani denies being party to the Iranian-sponsored deal and has warned outside powers against "imposing" anything on Iraq.
On Monday, he met the United Nations top official in Iraq, Jeanine Hennis-Plasschaert, to back her phased roadmap for tackling the crisis.
The plan calls for electoral reforms within two weeks followed by constitutional amendments and infrastructure legislation within three months.
On Friday, Sistani urged lawmakers to "work quickly to pass a fair electoral law that would restore people's faith in the electoral process". "Passing a law that does not provide this opportunity to voters would not be acceptable or useful," he said.
Parliament received a draft of a new electoral law this week but has yet to begin debating it.
A source with close ties to the Shia religious leadership told AFP that Iranian delegates had tried to deliver a letter to Sistani asking him to back the government and tell protesters to leave the streets.
Sistani "refused to answer the letter or even receive them," but he did meet with Soleimani, the source said.
"Qasem Soleimani heard some tough words from the marjaiyah about the Iranian role in the Iraqi crisis," he added.
The revered cleric is usually much less involved in politics, said Carnegie senior fellow Harith Hasan. "That is why his latest words on the protests revealed how seriously he perceived the current situation in Iraq," Hasan said. "By more clearly siding with the protesters, Sistani made one of his boldest moves yet, the outcome of which may determine the balance of power within the Shia community and Iraqi politics for years to come."