The number 52 which calls at stops in Woodhouse, the city centre and Hillsborough is regarded as a main commuter route used by people travelling to work, parents dropping off their kids at school and students attending the universities.
Passengers raised concern that this would be 'disastrous' as existing services would not be able to take on the extra load meaning commuters could be left stranded at stops watching buses drive past.
But Matt Davies, managing director of Stagecoach Yorkshire, defended the move today and said: "The service changes to the Sheffield bus network, scheduled for September, are designed to reflect current travel patterns, improve travel options and allow us to work together to tackle congestion in the city.
“Whilst the 52 service remains one of the busiest routes in Sheffield, fewer customers use the service between Crookes and Hillsborough."
Mr Davies highlighted how the company has invested millions of pounds across the wider network to improve passenger experience.
He said: "Buses for Sheffield is reinvesting over 40 hours per week of resource back into the bus network to meet demand and improve punctuality on the X1.
“First South Yorkshire and Stagecoach Yorkshire have invested £9 million in 44 new buses this year for Sheffield.
"Stagecoach recently extended the route of their gold service X17 between Sheffield, Meadowhall and Barnsley to meet increased customer demand.
"The eight new double decker gold buses mean passengers can benefit from free on-board wi-fi, luxury e-leather, high-back seats and ambient lighting.”
The response comes after one passenger - a 47-year-old mum of Lower Walkley who uses the service daily to take her kids to school - called for a re-think over the changes last week.
She claimed that the new route means about 10 stops will be missed on the 1.2 mile stretch between Crookes and Hillsborough.
The route will still be served by the First service 52a between Wisewood, Hillsborough, the city centre and Woodhouse.
In a message on their website, First said the changes have been introduced to "assist with punctuality."