Tens of millions of Americans who work at companies with 100 or more employees will need to be vaccinated against COVID-19 by Jan. 4 or get tested for the virus weekly.
The new government rules were issued Thursday.
The Occupational Safety and Health Administration says companies that fail to comply could face penalties of nearly $14,000 per violation.
The new requirements were first previewed by President Joe Biden in September.
They will apply to about 84 million workers at medium and large businesses.
It is not clear how many of those employees are unvaccinated.
A senior administration official says the agency would target companies if it gets complaints.
Republican governors, lawmakers and attorneys general already are forming a wall of opposition to the plan.
They have adopted laws to exempt employers in their state, promised lawsuits and in some cases are ensuring that workers who are fired for refusing a vaccine will have access to unemployment insurance.
They question the constitutionality of the federal regulation, saying the federal workplace safety agency does not have the power to impose vaccine mandates.