Gleneagles hotel is closing until February after Perth and Kinross moved into level three of coronavirus restrictions.
The five-star hotel and golf resort in Auchterarder will close to guests from Friday until January 31.
Its golf courses and membership facilities – including the clubhouse, equestrian school, shooting and fishing school and health club – will remain open, subject to government regulations.
The hotel said it plans to protect all jobs during the closure and bring all staff back for the February reopening.
The closure is the hotel’s second during the Covid-19 pandemic, with doors having only reopened in July.
Under the coronavirus restrictions, tourists should not travel into or out of Level three areas.
Conor O’Leary, Gleneagles’ managing director, 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.
“The support and well-wishes we’ve received over the past eight months, including our four-month closure period earlier this year, have been truly humbling and we look forward to welcoming back our hotel guests from February 1, subject to government regulations.”
The Scottish Tourism Alliance said the closure is “no surprise” given the new Covid-19 restrictions.
Chief executive, Marc Crothall, 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.”
Mr Crothall added he spoke to First Minister Nicola Sturgeon on Wednesday to outline his concerns.