Brussels on Friday said it had launched legal action against Slovakia over changes to the country's constitution that see national law take precedence over European Union law.
The amendments were passed in September as part of a move to limit the rights of LGBTQ people, which nationalist Prime Minister Robert Fico called a "historic step".
Fico, who returned to power in 2023, has often been at odds with the EU's executive, the European Commission.
He has faced a series of protests at home over his policies, and Europe's top rights organisation warned about the anti-LGBTQ amendment.
The specific change targeted by Brussels stated that Slovakia's "sovereignty" regarding "cultural and ethical questions" should override EU law.
"This contravenes the principle of the primacy of EU law, which is a fundamental element of the EU legal order," the EU said.
The commission said it had sent a letter of formal notice to Slovakia over the start of the legal action, known in EU jargon as an infringement procedure.
Bratislava now has two months to reply to the letter.
Fico, a supporter of US President Donald Trump, defended the constitutional amendment, saying Slovakia had the "right" to make it.
"I can confirm 100 percent that there will be no change," he told reporters.
The broader amendment passed by Slovak lawmakers said the country "recognises only two sexes, male and female".
"Sex cannot be modified except for serious reasons, according to procedures that will be established by law," it continued.
The amendment also only authorises adoption for married couples, with rare exceptions.
Fico has previously hailed the changes as "the best response to the collapse of Western societies, where progressivism and liberalism have been given priority".