The advertisement, by the Coalition for Marriage, features three women concerned about what their children will be taught in schools.
"The school told my son he could wear a dress next year if he felt like it," one says.
"Kids in Year 7 are being asked to role play being in a same-sex relationship," another says.
The Coalition for Marriage, the key organisation behind the 'no' campaign, said in a statement, the "plebiscite is a referendum on consequences".
"Removing gender from our marriage laws means removing gender from our classroom.
"This ad will play an important role in helping Australians understand that saying 'yes' to gay marriage would mean saying 'yes' to radical gay sex education in schools."
But The Equality Campaign has slammed the advertisement, describing it "dishonest", and saying that it aims to divide Australians on the issue.
“The people behind this ad know that Australians believe everyone should have the same opportunity to marry, so they are deliberately resorting to misleading people,” Tiernan Brady, The Equality Campaign's executive director said.
"Sadly, the Australian people will now hear a daily dose of red herrings and dishonesty served up by a huge bucket of cash.
"We would remind those behind this ad and all who engage in this national conversation that words can inflict real damage to people.”
The advertisement will run on all of the main commercial networks and pay-TV stations, and is also supported by the Australian Christian Lobby.
The Australian Bureau of Statistics will run the postal survey going to every enrolled Australian from next month, to be returned by 7 November.
A result is expected on 25 November.