Those travelling from Spain and its islands into the United Kingdom, including Northern Ireland, will now be required to quarantine for 14 days.
The government said the decision to introduce a quarantine period has been taken following medical and scientific advice.
The new regulations took effect from midnight last night.
Therefore anyone arriving after that time must self-isolate.
Speaking about the changes, 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.
“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.”
Further information is available on nidirect https://www.nidirect.gov.uk/articles/coronavirus-covid-19-countries-and-territories-exemptions
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