The former cabinet minister and Liberal frontbencher lost the top spot to outspoken backbencher Alex Antic.
The change has reignited debate about the treatment of women in the Liberal Party, as it struggles to increase representation in its parliamentary team.
Senator Antic angered Liberal Party colleagues during the pandemic for his staunch opposition to vaccine mandates and for voting with One Nation against the then-government's position.
Senior Liberal Simon Birmingham is among those who have expressed their support for Senator Ruston. (Lukas Coch/AAP PHOTOS)
He's since come out on the attack saying "the gender card" was being played following pre-selection.
His move was purely symbolic as second spot on the ticket is essentially locked in for the Liberals.
While Senator Ruston has kept quiet since the weekend's pre-selection stoush, senior Liberal Simon Birmingham expressed his support for her on Tuesday.
But he also acknowledged party members were entitled to have their say.
"The thing about democratic processes is you don't always get to like the outcome," he said.
"I was and am a strong supporter of Anne Ruston, she is a dear friend.
"I know that she will and continues to have a big role to play and that she will keep doing so for years to come."
The Liberals have been under pressure to pre-select more women in winnable seats to increase their representation in caucus.
MP Melissa McIntosh prevailed after a pre-selection battle in her western Sydney seat of Lindsay. (Mick Tsikas/AAP PHOTOS)
Victorian senator Jane Hume was one of the first of Senator Ruston's colleagues to publicly declare her support, branding the decision to put Senator Antic ahead of her a mistake.
Senator Hume also faced relegation if she did not accept second place on the Senate ticket behind Victorian colleague James Paterson, as power brokers would have pooled numbers to force her off or bump her down, Liberal sources said.
Recently promoted Liberal MP Melissa McIntosh faced a serious pre-selection battle in her western Sydney seat of Lindsay, but pressure from leaders has since forced her opponent to drop out.
Former minister Karen Andrews also pushed to have a woman succeed her in the safe Gold Coast seat of McPherson when she retires at the next election but to no avail.
If the Liberals maintain the same number of seats in their Queensland stronghold, there would only be one woman from the state if Ms Andrews' successor is a man.
