Animal welfare protesters claim three cows died on board a live exports shipment because the vessel was allowed to sail in "severe weather conditions".
Kent Against Live Exports (Kale) said the cattle were killed when the vehicle carrying them tipped onto its side in the ship's hold.
The livestock was being shipped from Dover to Dunkirk early on Thursday.
Kale said the sailing should have been prevented under animal welfare directives. An inquiry is under way.
The group said it contacted the State Veterinary Service (SVS) at about 0320 GMT on Thursday, saying there were "utterly terrible" weather warnings and pleading for the ship to be held at Dover.
But Kale said the Welfare of Animals (Transport) Order 1997 put the judgement at the discretion of the master of the vessel, who in this case had "decided to continue with the sailing".
But it resulted in an "unnecessary tragedy", Kale said.
Following the discovery of the cattle deaths at Dunkirk, the SVS said a full investigation would be carried out alongside its French counterparts.