Labor senator and leading "yes" vote advocate Pat Dodson warned that a "no" vote "will take us backwards".
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The senator's renewed push came as Opposition Leader Peter Dutton sought to fight complacency among Voice opponents amid concerns many may neglect to vote or vote informal.
In an apparent swipe at the anti-Voice slogan urging that "if you don't know, vote no", Senator Dodson told the National Press Club that voting "no" was "not a neutral matter".
Speaking remotely from Broome on Wednesday, where he is recovering from cancer treatment, the senator said that "voting 'no' is to say 'no' to the recognition of the Aboriginal and Torres Strait Islander peoples and to deny them a rightful place in our constitution".
"Don't vote 'no' because 'no' takes you nowhere," he said.
Senator Dodson said the referendum was the most significant vote people would make "for a very long time".
"The truth of our integrity as a nation is what is at stake here," he said.
Elsewhere, Mr Dutton - though campaigning for the "no" case - agreed, saying that "this is the most important vote that you will cast in your lifetime".
Reacting to concerns that the "no" vote's consistent lead in recent polls may encourage many anti-Voice supporters not to turn out, Mr Dutton cautioned against complacency, saying that "this vote's not won by any stretch of the imagination".
"If you're a 'no' supporter and you think a 'no' vote is going to get up, don't take it for granted. You've got to get out and vote," he said.
Earlier on Wednesday, Prime Minister Anthony Albanese greeted ultra marathon runner and former Liberal MP Pat Farmer at Uluru as he ended his 14,500-kilometre run in support of a "yes" vote.
The Prime Minister expressed his "admiration and awe" for Mr Farmer, saying he had "provided an inspiration for Australians".
"There are so many Australians out there giving up their time to advance this nation in a positive way," Mr Albanese said. "But no one has done more that this bloke."
"What he has done in running 14,500 kilometres [is] to ask his fellow Australians to just walk a few metres, walk into a polling booth and write 'yes' on the ballot paper," he said.
An emotional Mr Farmer, who started his run six months ago, urged Australians voting at the referendum to "not take a backward step".
"I want to encourage all of you to vote yes, [to] move forward together as one".
Mr Albanese, also speaking on KIIS FM, dismissed polls suggesting overwhelming support for the "no" case at the referendum, including a Channel 7 survey indicating 72 per cent intended to vote no.
The Prime Minister said the "yes" proposition was "pretty straightforward".
"We're recognising a fact and then the form of recognition is that we're just having an advisory committee. That's all it is," he said.
"It doesn't change the way that decisions are made, it doesn't change the power of the Parliament or the power of government. It's simply a non-binding advisory committee, so we can listen to people."
The Prime Minister said the "no" case was "where we are now. We are living in 'no'. We know it's not working".
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Senator Dodson said that the country will need to seriously reflect on its actions and decide how it will engage with First Nations people if the "no" campaign succeeds at Saturday's vote,
"We need to look in the mirror and say who the hell are we? What have we done? And what are we going to do about it?," he said.
"It's not just a question for the Parliament. It's a question for the Australian people as it will be for the government.
"The 'no' campaign will take us backwards. That to me is the sorry part of the outcome for the 'no' campaign ... You still need to engage with the Aboriginal people. They're not going away, they're not disappearing. They may be a bit more stroppy than I am when they come to deal with you."