The so-called “travel corridor” with Spain was closed from midnight.
The decision comes after Spain reported more than 900 new daily infections for the past two days.
The decision means those coming back from the popular holiday destination, including its islands, will have to self-isolate for two weeks upon their return to Northern Ireland.
The move will come as a surprise to returning holidaymakers who left when Spain was part of the safe travel list.
In a statement, the Department of Health said: “The decision to introduce a quarantine period has been taken following medical and scientific advice. The new regulations will take effect from midnight, therefore anyone arriving after that time must self-isolate.”
Health Minister Robin Swann said: “The decision to remove Spain from the exempt list was not taken lightly.
“I have always said I will move swiftly and take the necessary action to ensure our citizens are protected.
“I have spoken with my counterparts across the UK today and we agree that the recent upsurge in new positive cases in different parts of Spain are a cause for concern.
“As of midnight tonight, anyone returning or visiting Northern Ireland from Spain, including its islands, will be required to quarantine for 14 days.
“I will continue to monitor the situation in Spain and other countries, and make the changes to the regulations when necessary.”
There are five flights arriving into Belfast International Airport from Spain on Sunday.
They include flights from Malaga, Barcelona, Alicante, Palma de Mallorca and Ibiza.
There are also three flights departing the airport to Spain, with hundreds of holidaymakers from Northern Ireland.
The flights are being operated by EasyJet, Jet2 and Ryanair.