More than 70 of the 75 staff went on strike outside the Eastern Avenue jobcentre at Manor Top.
Union representatives and a local MP joined the strike which crippled the centre for the day.
Welfare payments were paid in advance and about 70 jobseekers allowance interviews were excused to ensure no customers were affected by the industrial action.
Workers from Annesley in Nottinghamshire joined the Sheffield staff on the strike. That centre has also been earmarked for closure.
Sheffield staff member Clare Goonan applauded her colleagues for 'speaking with their feet' and walking off the job.
Mrs Goonan, who has worked at Eastern Avenue for 12 years, said the 6,000-7,000 people who used the service would be the ones who lost out if it closed.
She said the staff jobs had been guaranteed. They will be moved to other centres.
Taking a bus into the city could have a serious impact on clients' finances and mental well-being, Mrs Goonan said.
A CityBus ticket with First South Yorkshire costs Â£15 per week.
"And when you're on Â£73 per week, that's a lot," Mrs Goonan, who's 41, said.
Customers with mental health issues will have trouble making their way into town.
"It could take them weeks of confidence-boosting," Mrs Goonan said.
"In town, they're less likely to get a personal service," Mrs Goonan said.
Sheffield Labour candidate Louise Haigh and Public and Commercial Services Union representative Charles Law joined picketers today.
Mr Law said it would be an 'absolute scandal' if the service closed.
"It's a key service for this area of Sheffield," he said.
"What we're on strike for is to maintain this community based service."
Mr Law is 'very optimistic' the centre would stay open.
"We've built a really good campaign," he said.
That campaign has included leaflet dropping and petitions, and a public meeting.
"The local community has supported it, and the local MP is supporting it," Mr Law said.
Another strike is planned for Monday, June 12 at the centre,
The DWP has proposed to close 78 jobcentres.
Closure of the Eastern Avenue service would leave five centres in Sheffield: Chapeltown, Hillsborough, West Street, Cavendish Court and Woodhouse.
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