The MTR Corporation has told its staff they won't be allowed to enter the company's premises if they haven't had at least one dose of a Covid vaccine by February 23.
The rail operator said on Sunday that the requirement applies to all staff members, except those who are medically exempted.
It will not accept regular testing as an alternative, it added.
The company said the move is aimed at protecting staff and passengers in view of the coronavirus situation in Hong Kong.
It also said that staff eligible for a booster should get one as soon as possible.
Chief Executive Carrie Lam earlier announced that vaccine mandates will be tightened on February 24. On top of entertainment venues, the requirement for customers to have received jabs will be extended to include restaurants, schools, hotels, libraries, museums and other places.
Lam also said that the government would urge statutory bodies to require their staff to get vaccinated.