The crisis in Ukraine has made the planned pipeline a new focus of tensions between Moscow, Brussels and Washington. EU member Bulgaria earlier this month suspended work on building its section of the multi-billion-euro (-dollar) project following pressure from the EU and the United States.
"This project is not aimed against anyone. This project is being done in the interest of energy stability in Europe as a whole," Putin told the Vienna Chamber of Commerce.
"We never work against anyone ... (South Stream) should not be politicised. Both Russia and our European partners need this project."
In reference to the Ukraine crisis he said that the pipeline, running from Russia under the Black Sea and through the Balkans to Italy and Austria, would "diversify energy supplies" for the European Union.
At present Europe gets around a third of its gas from Russia, and half of this comes through Ukraine, which has been through several months of turmoil. Previous disputes between Moscow and Kiev have hit supplies to the bloc.
"Implementing the South Stream pipeline as soon as possible will contribute to energy security and diversify energy supplies. This is of particular importance in the context of the current developments in Ukraine ... We have often had to confront crises like this," Putin said.
His comments in Vienna came after Gazprom inked a deal with Austria's OMV approving Austria's section of the pipeline, which Putin said would make Austria Europe's biggest hub for Russian gas.
The European Commission has called on all 28 member states to stand united in resisting pressure from the Kremlin over the project, saying the contracts Gazprom has signed on the pipeline breach the bloc's competition rules.
Moscow says the pipeline will ensure European energy security and has accused Brussels of pressuring Bulgaria into suspending work in order to seek revenge over the Kremlin's alleged role in supporting separatists in eastern Ukraine.
But South Stream has also exposed divisions within the bloc, with several states that depend on Russian gas transported via Ukraine supporting it, together with countries on the pipeline's route.