Preston Bus has confirmed that its 280 service, which winds its way from Preston to Clitheroe and onto Skipton, will be withdrawn on Saturday, June 15.
The bus operator took over the rural route in March 2016 after French firm TransDev opted not to renew its contract.
But the route, which links Lancashire with Yorkshire, has not proved sustainable for the bus operator.
The route currently serves rural communities including Mellor Brook, Copster Green, Billington and Whalley en route to Skipton.
Lancashire County Council has put the service out for tender and is seeking bids from local bus operators interested in continuing the service.
A spokesman for Lancashire County Council said: "The withdrawal of Service 280 is a commercial decision taken by Rotala Preston Bus.
"We are aware this will have an impact on passengers and are currently working with other bus service providers to establish if a sustainable replacement service can be provided."
A spokesman for Preston Bus added: "Preston Bus has de-registered commercial operation of Service 280 from Monday, June 17.
"But the service is likely to finish after its last timetabled journey on Saturday, June 15.
"Lancashire County Council has issued a tender, inviting bids from local bus operators to ascertain the potential cost to replace the withdrawn commercial service.
"However, at this moment in time there is no guarantee a contract will be awarded.
"We’d like to thank the dedicated team of drivers on the 280 for their sterling efforts over the years. Reports from customers have been consistently outstanding.”
The bus firm said assured customers that any unused portions of its Gold EasiWeek and Gold Easi28Day tickets will be refunded.