Tram services between Tinsley/Meadowhall South and Meadowhall Interchange are to be suspended for two weeks because of nearby roadworks.
Services will stop from Monday and will not resume until Monday, January 27.
Replacement buses will run between the two stops at the same frequency as trams and tram tickets will be valid on the services.
Buses will be scheduled to meet arriving trams.
Tonight, between 9.30pm and 12.15am a bus replacement service will be in place between Attercliffe and Meadowhall Interchange.
The disruption is due to preparatory work needed to construct a new section of road under Tinsley Viaduct. The road, which forms part of the £30 million Bus Rapid Transit scheme, will eventually allow for quicker journeys between Sheffield and Rotherham, avoiding Junction 34 of the M1.
It is due to open in 2015, after the approval of funding from the Department for Transport last October.
David Young, for South Yorkshire Passenger Transport Executive, said: “We apologise to customers for the disruption. The work is essential to allow construction to start soon on the link road under Tinsley Viaduct.”
The BRT scheme will link Sheffield and Rotherham via a high-frequency, limited -stop bus service.