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.
One passenger branded the move 'disastrous' and claimed existing services would not be able to take on the extra load meaning commuters could be left stranded at stops while too full buses drive on.
Although the route will still be served by the First service 52a between Wisewood, Hillsborough, the city centre and Woodhouse - this will only be every ten minutes between Crookes and Hillsborough whereas it used to be every five when both the 52 and 52a were running.
Passengers called for First to increase the frequency of the 52a to every five mins in the morning and evening to compensate for loss of the 52.
Allan Riggall, head of commercial at First South Yorkshire, said: "The September changes will mean that a five minute service will still remain between Woodhouse and Crookes, and a 10 minute service will still operate between Crookes, Hillsborough and Wisewood.
“A 10 minute service between Hillsborough and Crookes is sufficient for demand.”
This comes after Matt Davies, managing director of Stagecoach Yorkshire, said the change was introduced because while the 52 is "one of the busiest routes in Sheffield, fewer customers use the service between Crookes and Hillsborough."
She said: "There is a big population down here and a lot of people that will not be served as well."