The Gleneagles hotel in Perthshire is to close for 11 weeks due to tighter coronavirus rules coming into force in the area.
Managing director Conor O'Leary said the team had taken the "very difficult decision" to shut down when Perth and Kinross moves from level two to level three restrictions on Friday.
The hotel will close to guests from 13 November until 31 January 2021, though its golf courses and membership facilities will remain open.
A spokesperson told The Caterer Gleneagles is aiming to protect all staff jobs and will furlough affected employees until February.
The hotel shut for four months earlier this year as part of the national lockdown.
O'Leary said: "Having worked so hard to provide a safe environment across our estate, we are saddened by this development, especially in the lead-up to Christmas – the highlight of our calendar for both team and guests. However, we're committed to taking the additional measures necessary to protect the health and wellbeing of everyone at Gleneagles, and to play our part in minimising Covid-19 cases in the region."
Under Scotland's level three restrictions pubs and restaurants must close at 6pm and are banned from serving alcohol. People cannot meet in groups larger than six from more than two separate households.
Marc Crothall, chief executive of the Scottish Tourism Alliance, said news of the closure came as "no surprise" to the trade.
He said: "I have been having conversations with hoteliers the length and breadth of the country and many are doing similar. The costs of remaining open and providing a total experience which the customer expects, with limitations and uncertainty, are too high.
"While furlough is a welcome support for staff who would almost certainly be without a job right now in many businesses had it not been extended, it offers no direct financial benefit to businesses and a much greater degree of financial support is needed immediately to enable those both open and closed to remain solvent and bounce back when we are through the crisis period and into recovery."
Crothall added that he had raised the issue on a private call with first minister Nicola Sturgeon and hoped additional support would be announced ‘in the very near future'.
The Scottish Hospitality Group is calling on the Scottish government to alter the tier restrictions or publish scientific evidence behind its curbs on trading hours.