Cigarettes are now prohibited at Bandyup Women's Prison, the first custodial site in WA to go smoke-free.
Cigarettes and tobacco products are no longer on sale to prisoners at the facility and have been listed among contraband items at the site.
Smoking is also banned for all prisoners, staff, visitors and contractors at the Middle Swan prison.
"The McGowan Government is committed to providing a healthy and safe environment for people working in, visiting, and in the care of custodial facilities," Corrective Services Minister Bill Johnston said.
"We are giving prisoners an opportunity to quit and have a fresh start.
"Western Australia is lagging behind other Australian jurisdictions where smoking has already been successfully phased out in prisons.
"This pilot will inform the staged adoption of smoke-free policies at all facilities in the State."
The Cancer Council WA and Australian Council on Smoking and Health are supporting the move and will provide ongoing assistance to prisoners and staff at the site.
Ahead of the pilot, smoking reduction strategies were implemented, including support groups, education and health programs, and recreational activities.
Nicotine replacement therapies are being provided to prisoners and staff as an interim support measure.
Prisoners and staff found smoking may face disciplinary action, while visitors smuggling contraband could face criminal prosecution and a ban on visiting the site.