The Bob Barker, which was once a Norwegian whaling ship, steamed out of Hobart on the mission which aims to harass the Japanese fleet as they harpoon the giant animals and prevent them from taking their full quota.
Captain Peter Hammarstedt said it was his ninth campaign protecting whales, which have at times included high-seas clashes between the conservationists and the Japanese.
"The Japanese whaling fleet intends to kill 1,035 whales of which 50 are endangered fin whales and 50 are endangered humpback whales, the very same whales that frequent the shores here off Australia," he said.
"Our intention is to once again intercept the Japanese whaling fleet in the Southern Ocean whale sanctuary and to do everything that we can."
Sea Shepherd Australia said its two other boats the Steve Irwin and Sam Simon left Melbourne on Wednesday for the annual campaign it took over from the US-based Sea Shepherd Conservation Society in late 2012.
The Australian government, which strongly opposes Japanese whaling, is expected to announce later this week that a Customs vessel will monitor the hunt.
"The government's commitment to monitoring in the Southern Ocean remains undiminished," a spokesman for Environment Minister Greg Hunt said in an email.
But the left-leaning Greens said the purpose-built Customs vessel that would be used was nowhere near the Southern Ocean, instead in waters north of Australia on a border protection mission.
"It is purpose-built for the Antarctic, it is ice-rated, it is sitting off topical waters on Christmas Island," said Greens Senator Peter Whish-Wilson.
"I've got a photo of it during the week sitting in tropical waters. What's it doing there?"
Australia wants Japan's annual whale hunt in the southern hemisphere summer to stop and has taken the matter to the UN's top court the International Court of Justice. A decision is expected in early 2014.
Japan says it conducts vital scientific research using a loophole in an international ban on whaling, but makes no secret of the fact that the mammals ultimately end up as food.
