Rex has today announced that all of the airline's customer-facing staff must be fully vaccinated against COVID-19 by November 1.
This includes employees working at check-in and all pilots and cabin crew across its regional and domestic networks.
Unvaccinated frontline staff will be offered non customer-facing roles where available.
Office staff who have not been vaccinated will be required to wear a mask while at work.
Rex deputy chairman John Sharp said the compulsory staff vaccination policy meant passengers would be travelling in the safest possible circumstances.
"We have a duty of care to both our passengers and staff to provide the safest possible environment," Mr Sharp said.
"As we provide an essential service operating to regional centres and remote communities throughout Australia, it is incumbent upon us to do whatever we can to help those residents remain safe and healthy."
Mr Sharp said Rex had always been the leader in keeping its passengers safe and healthy.
"As early as April last year, we mandated the wearing of masks onboard. The other carriers did not have the courage to do so until the Federal Government had to step in six months later to make this a regulatory requirement," he said.
"Similarly, today Rex will be leading the industry by having all its crew and frontline staff fully vaccinated by November."
The move follows consultations with unions, workplace health and safety representatives and staff.
Prior to the consultations, Rex conducted a survey of all its staff, to which 90 per cent responded.
In the survey results, 59pc said they were already fully vaccinated; 31pc had either received their first vaccination or had booked an appointment to be vaccinated; 8pc were not sure about vaccination; and 2pc were opposed to vaccination for medical or other reasons.
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