Greens leader Richard Di Natale has called the three senators 'cowards' for walking out.
Conservative senators have walked out of parliament as Greens leader Richard Di Natale called for stronger rules to police sexism in the upper house.
Nationals senator Barry O'Sullivan, Liberal Democrats' David Leyonhjelm and independent Fraser Anning left the chamber after calls from all sides of politics to lift the standard of debate.
"We are allowing women to be demeaned in this chamber," Senator Di Natale told parliament on Wednesday.
"They stand on all sides of this chamber but predominantly they are those people who are walking out right now."
It came a day after Senator O'Sullivan was labelled a "pig" by Senator Di Natale for saying the Greens' Sarah Hanson-Young "had a bit of Nick Xenophon in her".
The Greens leader was suspended from parliament after refusing to withdraw the comment, saying he was taking a stand against constant sexist abuse towards female senators.
Senator Di Natale said men were openly and brazenly shaming, insulting and harassing female MPs in the upper house.
"It reinforces that culture that leads to 72 women being murdered by their partners this year," he said.
Senator O'Sullivan, who claims he was talking about Senator Hanson-Young's absence from an inquiry, shook his head during speeches from Labor Senate leader Penny Wong and Senator Di Natale.
Senator Wong said people expected a line to be drawn between acceptable political debate and personal smears and innuendo.
"We saw those expectations trashed yesterday by Senator O'Sullivan - and he is not the first person in this chamber to do so - by his engaging in deliberately offensive personal remarks," Senator Wong said.
Senate President Scott Ryan said unparliamentary, offensive epithets and abuse had no place in the chamber.
"This is rightly a place of vigorous debate, but personal abuse has no place, particularly if it targets personal attributes such as race or gender," he said.
Government Senate leader Mathias Cormann said there needed to be a respectful approach to the battle of ideas.
"There is always a requirement and a responsibility on all of us to engage in the debate in a way that is appropriately robust but also appropriately respectful," Senator Cormann said.
The coalition and Labor backed a proposal from the Senate president to stamp out tactics which promote divisive debates that have seen tensions boil over recently.