The agency is alleging violations of the Jones Act, a law requiring vessels that carry goods between two US points to be American-made and flagged.
Responding, Kloosterboer International Forwarding and Alaska Reefer Management filed a 35-page complaint in US District Court in Anchorage.
The two companies are suing to stop the penalties, contacting shipowners, cold storage operators, and trucking and fishing companies to transport frozen seafood.
Penalty notices have been issued since the Canadian rail route is used in the process, even though the agency has supported the route in its published interpretative rulings, according to the complaint.
The companies declared the notices threaten a long-established supply chain and jobs in Alaska and the Lower 48.
'Penalty notices effectively have shut down a critical shipping route that - for over 20 years - has been approved by customs as complying with the Jones Act, and which is essential to the delivery of frozen seafood to consumers, fast food chains, and school lunch and food bank programs throughout the United States,' said the complaint.
The penalties for Kloosterboer total $25 million, with numerous other companies in the supply chain have also received notices totaling over $325 million.