Japan aims to take more than 300 whales in its "scientific whaling" programme before the hunt ends next year.
The International Court of Justice (ICJ) ruled last year that Japan's whaling in the Southern Ocean should stop and an International Whaling Commission (IWC) panel said in April that Japan had yet to demonstrate a need for "lethal sampling".
But Tokyo, which had vowed from the start to resume its "scientific whaling" programme from the 2015/2016 season, retooled its hunt plan to cut the number of minke whales it intends to take to 333, down by two-thirds from previous hunts.
"Last year, regrettably, the ICJ made its ruling and we were unable to take whales," said Tomoaki Nakao, the mayor of the western city of Shimonoseki that is home to much of Japan's whaling fleet and part of Prime Minister Shinzo Abe's election district.
"There's nothing as happy as this day," he told the fleet's crew at a ceremony prior to their departure.
Shortly before noon the ships sailed away under a clear blue sky, with family members and officials waving from shore.
The hunt is expected to last until March.
Japan, which has long maintained that most whale species are not endangered and that eating whale is part of its food culture, began what it calls "scientific whaling" in 1987, a year after an international whaling moratorium took effect.
It says its whaling surveys are needed for information on how whales live, including what they eat, as well as population numbers.
The meat ends up on store shelves, although most Japanese no longer eat it.
Officials, including Abe, have long said their ultimate goal is the resumption of commercial whaling - a pledge Abe repeated in a message read at the pre-departure ceremony.
Australian Environment Minister Greg Hunt said in a statement that his country strongly opposes Japan's decision.
"We do not accept in any way, shape or form the concept of killing whales for scientific research," he added. (Writing by Elaine Lies; Editing by Michael Perry)