US Attorney Catherine Hanaway reached a settlement with online gambling company BetOnSports PLC that permanently bars the London-based company from accepting any bets from the US.
The lawsuit has been closely watched by the online gambling industry, which generates about US$6 billion annually in the US.
The settlement announced on Thursday ends a massive civil case Hanaway filed this summer. It does not affect a criminal case still pending against several BetOnSports employees, including Chief Executive David Carruthers, who remains under house arrest in a St Louis suburb.
Carruthers faces 22 counts of fraud and racketeering charges. His case is scheduled to go to trial early next year. BetOnSports fired him soon after he was arrested in July.
Hanaway spokeswoman Jan Diltz said the US Attorney's Office would not comment on the civil settlement because the criminal case is ongoing.
BetOnSports said in a statement that it had not admit wrongdoing by entering the settlement.
Spokesman Kevin Smith said about 20 percent of the company's revenue came from other countries, most of them in Asia.
"That will be the total focus of the company now," he said.
The settlement requires BetOnSports to open a toll-free telephone service to inform bettors how they can reclaim wagers pending before the suit was filed.
Smith said the hotline should be running within a week. He said he did not know how BetOnSports planned to refund customers.