A measure making it more difficult for foreign workers to be employed won 58 percent of the vote in a referendum on Sunday in the Italian-speaking canton of Ticino, which borders Italy.
Brussels said the vote would further complicate thorny negotiations over a national vote in early 2014 in which Switzerland voted for similar curbs, despite them violating the EU's free movement rules.
"The EU and Switzerland have been in intense talks for months now in order to find a solution on how to implement the Swiss popular vote on free movement in a way that respects obligations under the free movement agreement," said Margaritis Schinas, spokesman for commission head Jean-Claude Juncker.
"Yesterday's vote will not make the already difficult talks any easier," he told a daily briefing.
EU Commission President Juncker will visit Switzerland at the end of October to continue discussions in order to find "an agreement acceptable to both sides", said Schinas.
Switzerland is not an EU member but is signed up to the bloc's Schengen agreement, which allows passport-free travel and free movement of workers.
The referendum in Ticino was presented by the conservative Swiss People's Party. The vote's result still requires approval from the Swiss federal government.
However Bern is unlikely to look kindly at the Ticino vote, as it struggles to repair frayed relations with the EU and figure out how to apply the 2014 vote.
The EU is keen to take a hard line on Switzerland shirking its duties from bilateral agreements ahead of the bloc's negotiations with London over its departure from the bloc.
Pro-Brexit campaigners want Britain to limit migration from EU countries but keep access to the single market -- a combination that EU leaders have repeatedly warned is impossible.