From tomorrow (13 November), when the region moves from Tier 2 to Tier 3, the hotel will close for 11 weeks, reopening on 1 February.
While hotels are allowed to remain open under Tier 3 of Scotland's Coronavirus alert system, strict operating restrictions including a ban on alcohol sales must be followed.
Gleneagles, which is owned by hospitality group Ennismore, says that its golf courses and membership facilities will remain open for now, subject to Government restrictions.
The hotel adds that it plans to protect all jobs during the closure.
"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," says Conor O'Leary, Gleneagles' managing director.
"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 hotel closed for four months earlier this year as part of the national lockdown.
Gleneagles' announcement comes a day after the Scottish Hospitality Group (SHG) called on the devolved government to 'tweak' the tiered Coronavirus restrictions in a bid to help save thousands of jobs and prevent many business closures.
The industry body, which comprises nine of the country’s largest independent hospitality operators and collectively employ over 6,000 people, urged the Scottish Government to change operational rules in Levels 2 and 3 of the country's Coronavirus restrictions to allow hospitality venues to remain open until 10pm rather than have to close at 8pm or 6pm.
It has also demanded the Scottish Government publishes the scientific evidence behind its decision-making related to the restrictions placed on hospitality.
