John Coates, the chief executive of Bet365, said the donation was from his father Peter, the chairman and owner of the company and a long-standing Labour supporter, and was “not connected to his business”.
The government earlier this month agreed to allow internet gambling sites to advertise on television.
Mr Coates said the donation was not in any way linked to the new rules, which contained “not a lot of pluses” for his industry.
The original bill would have given gambling websites even greater freedom to advertise but the government had since rowed back, he said. The industry was made to sign up to a code of conduct banning it before the 9pm watershed apart from during sporting events.