The decision was announced following consultation with employees in the Barker’s Pool store which has been closed since the beginning of Lockdown 3.0 in January.
A spokeswoman said they would talk to each of the 299 workers about redeployment.
Patrick Duffy, head of the Sheffield branch, said: “John Lewis holds a special place in my and many peoples' hearts in Sheffield, so confirmation of our closure is very sad for the city, our Partners and our customers.
“It has been a great honour to be a part of a wonderfully dedicated team, who are so proud of the long and valued legacy of service we’ve held in Sheffield. I want to thank current and former Partners for all their hard work over the years - and for the memories that we have shared together.
“I also want to thank our customers for their loyalty, the joy they’ve brought to our shop and for the support they have shown for us in these past few months - we are very grateful and have been truly overwhelmed.”
The news comes as John Lewis continues to negotiate with Sheffield City Council over how much to pay for breaking its 20-year lease, signed in August last year.
Sheffield City Council spent years ‘bending over backwards’ to get the company to stay, culminating in the authority spending £3.4m buying John Lewis out of its lease and renting the building back on a much-reduced rate based on turnover.
But in March, the retailer said it did not plan to reopen the Barker’s Pool shop after lockdown. It was one of eight the firm said it ‘could not profitably sustain’.
More than 20,000 people signed a petition opposing the proposal.
In April, Sheffield MP Paul Blomfield met John Lewis bosses and said he did not understand why the company was closing.
He said then: “They said it was profitable when the firm signed the new deal with the council last summer but they were unable to say what had changed since then.
“They anticipated trade would not come back and there would be a permanent shift to online shopping. But they are making assumptions during a pandemic. It would have made sense to test them by reopening for a trial period.”