Tamara Kelekhsayeva, a spokeswoman for the leadership of the breakaway region, said on Monday the vote would be held on November 12 in a step that is likely to increase tensions between Russia and Georgia.
South Ossetia, a tiny region near Russia's southern Caucasian border, is a frequent source of tension between the two and fought a brief war in the early 1990s for independence from Georgia.
After that conflict, South Ossetia voted for independence in a referendum in 1992. It was not immediately clear why a new referendum was needed.
Kelekhsayeva said the question would be "whether you agree or not that South Ossetia should preserve its present status of an independent state and be recognised by the international community".
Georgia accuses Russia of supporting the rebel province's rulers, and its parliament in July accused Moscow of trying to annex the territory together with Abkhazia, another breakaway province of Georgia.
Only last week, Georgia accused the Russian military of being involved in an attempt by rebels to shoot down the helicopter carrying the country’s defence minister, Irakly Okruashvili.
Tension has risen since Mikhail Saakashvili was elected Georgian president in 2004 and pledged to re-unify his country.
Besarion Jugeli, one of the leaders of the United National Movement party, which has a majority in the Georgian parliament, said: "This is political absurdity as this referendum will have no legal force."
Russia has peacekeeping troops in South Ossetia, and while Moscow says they keep the two sides apart, Tbilisi complains that they side with the separatists.
"This is political absurdity as this referendum will have no legal force"
United National Movement party
Vladimir Putin, the Russian president, said on Saturday that if Serbia's breakaway province of Kosovo was given independence, then ex-Soviet regions seeking self-rule should be rewarded likewise.
Putin mentioned South Ossetia and Abkhazia, the Dnestr region that broke away from Moldova in the 1990s and Nagorno-Karabakh, a separatist region of Azerbaijan, according to people at the meeting.
Dnestr will hold a referendum on September 17 to confirm independence. The Organisation for Security and Cooperation in Europe (OSCE) has said it will not recognise the vote.