There will be "serious consequences" if the UK triggers Article 16, European Commission (EC) Vice-President Maros Sefcovic has warned.
Mr Sefcovic said the move would be "serious for Northern Ireland as it would lead to instability and unpredictability".
His comments follow a meeting with the UK's Brexit minister in Brussels over the protocol dispute.
Lord Frost said progress at the meeting was "limited".
He said gaps could still be bridged through intensive negotiations.
The protocol is the special Brexit deal agreed to prevent a hard border on the island of Ireland.
It keeps Northern Ireland in the EU's single market for goods and allows free-flowing trade with the EU.
But it also creates a trade border between Northern Ireland and Great Britain.
The EU has proposed measures to ease the checks and controls for goods crossing the Irish Sea.
But the UK is demanding fundamental reform and there is growing speculation it will trigger Article 16 - which allows parts of the protocol to be unilaterally suspended if they are causing serious difficulties - in the coming weeks.
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