Northern Irish agriculture minister Edwin Poots announced on Wednesday that border checks on food and agricultural goods coming into Northern Ireland from the rest of the UK would be halted at midnight.
"The UK Government should ensure respect for the international obligations into which it has entered. Reneging on the provisions of these agreements undermines the credibility and trustworthiness of our partners," a statement by leading EU lawmakers read.
The European Parliament said the contentious protocol would not be renegotiated since it was "sufficiently flexible" to solve all practical challenges.
The United Kingdom and the European Union signed the so-called Ireland protocol before Brexit designed to avoid a hard border between Northern Ireland and EU member Ireland but effectively creating one between parts of the UK, much to the discontent of Irish unionists.
Northern Ireland's First Minister Paul Givan has announced his resignation, effective Friday midnight, amid tensions in the governing Democratic Unionist Party.