Japan's whaling fleet left for the Southern Ocean last month, planning to kill 333 minke whales over a four-month period.
Its fisheries agency says the hunt is needed to study whale behaviour and biology, but critics say such lethal research is unnecessary and acts as a cover for commercial whaling.
"(We) jointly express... opposition to Japan’s continued so-called 'scientific' whaling in the Southern Ocean," the EU and its allies said in a statement.
"We remain resolutely opposed to commercial whaling, in particular in the Southern Ocean Whale Sanctuary established by the International Whaling Commission (IWC)."
In addition to the EU, the letter was signed by Argentina, Australia, Brazil, Chile, Costa Rica, the Dominican Republic, Ecuador, Mexico, New Zealand, Panama, Peru and Uruguay.
Japan is a signatory to the IWC's moratorium on hunting, but exploits a loophole that allows whales to be killed in the name of scientific research.
In 2014, the UN's International Court of Justice ordered Tokyo to end its regular hunt in the Antarctic waters, saying the project did not meet conventional scientific standards.
Japan cancelled its 2014-15 hunt, only to resume it the following year under a new programme -- saying the fresh plan had genuine scientific value.
The joint statement noted that the IWC ruled last year that the new research programme was not scientific and should be halted.
"Japan's decision to return to the Southern Ocean this year is contrary to the commission's requests," it added.
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