The checkpoint, near the town of Slavyansk, had been manned by separatists in and around the Donetsk region, where armed separatists have occupied administrative buildings for the past week.
A holiday truce had been declared by Kiev's interim government, which pledged not to use force to dislodge the separatists who have called for secession of this predominately Russian-speaking region.
Yuri Zhadobin, who coordinates the pro-Russia unit manning the checkpoint, told The Associated Press he was with about 20 men celebrating Easter when unknown men drove up in four vehicles and opened fire about 3 a.m.
"We began to shoot back from behind the barricades and we threw Molotov cocktails at them," Zhadobin said. Two of the vehicles caught fire and the attackers fled in the other two, he said. Some of his men were wounded and one later died in the hospital, he said.