People in need of overnight emergency hospital treatment will no longer be taken by ambulance to Cheltenham.
Instead, in a change that has been criticised by campaigners and local MPs, "blue-light" patients will be taken straight to Gloucester, eight miles (13km) away.
Cheltenham's casualty department will remain open to walk-in patients.
One reason given for the change, taking effect between 20:00 and 08:00, is a shortage of emergency department staff.
Dr Tom Llewellyn, an emergency medicine consultant, said: "Our priority is to ensure that the sickest patients are seen by very skilled specialist staff when they need to be.
"The changes have been developed for quality and safety reasons, which includes the availability of experienced emergency medicine doctors."
The Gloucestershire Hospitals NHS Trust estimates the change will affect about 16 patients a night who arrive by ambulance.
The proposal has been heavily criticised by campaigners, the MPs for Cheltenham and the Cotswolds, and the town's chamber of commerce.