Tony and Julie Walker have now given up the lease on the popular converted double decker, which has sat on the layby between Leeds and Bramham for decades.
Leeds' famous Red Bus Cafe has now closedThere was an outpouring of disappointment on the business's Facebook page when the Walkers announced they had ceased trading over the weekend.
They admitted that the venture had not been able to turn a profit since taking it over 15 months ago due to the high running costs.
The owners of the site now hope to speak to potential new tenants interested in renting the cafe.
"A really, really sad day after all the hard work the family and I have put in. Anyone thinking of renting this place needs to speak to me first.
"We’ve been here since June last year, and it’s been a crazy 15 months. We’d like to thank all our customers, regular and not, for the laughs, patience and loyalty.
"We close our doors for the last time this afternoon, and wish any successors the best of luck."
The part of the layby where the bus stands is on private land owned by the trustees of the Viscounts Pollington.
The bus has had several changes of management in recent years.
The vehicle itself was built in 1968 and was in service in the north-east during the 1970s, under the ownership of Newcastle Corporation.