"The European Union and the United States of America are concerned about the continued tense situation in the north of Kosovo," they said in a joint statement.
"We call on everyone to exercise maximum restraint, to take immediate action to unconditionally deescalate the situation, and to refrain from provocations, threats, or intimidation," they said, after Kosovo closed its biggest border crossing with its northern neighbor.
The EU and US said they were working with Serbian President Aleksandar Vucic and Kosovo's Prime Minister Albin Kurti to seek a political solution to one of the worst flare-ups in years in northern Kosovo, where ethnic Serbs make up the majority.
Northern Kosovo has been on edge since November when hundreds of ethnic Serb workers in the Kosovo police as well as judges and prosecutors walked off the job.
They were protesting a controversial decision to ban Serbs living in Kosovo from using Belgrade-issued vehicle license plates, a policy that was eventually scrapped by Pristina.
On December 10, protesting ethnic Serbs erected barricades that effectively sealed off traffic on two border crossings.
Several shooting incidents targeted Kosovar police and international peacekeepers, and Serbian armed forces went on heightened alert.
Late Tuesday, dozens of demonstrators on the Serbian side of the border used trucks and tractors to halt traffic leading to Merdare, the biggest crossing between the neighbors, a move which forced Kosovo police to close the entry point on Wednesday.
In their statement, the EU and US said they had welcomed assurances from Kosovar officials that there was no arrest list for ethnic Serbs who may have taken part in peaceful protests and erecting the barricades.
"At the same time, rule of law must be respected, and any form of violence is unacceptable and will not be tolerated," they said.
"We also expect Kosovo and Serbia to return to fostering an environment conducive to reconciliation, regional stability, and cooperation for the benefit of their citizens," they said.