The incident occurred around 2:30 p.m. local time (1230 GMT) at a checkpoint fortified by Ukrainian forces near Volnovakha town, said Elena Davydovska-Malutina, a spokesperson for the Donetsk Regional Administration.
She said the shell came from the insurgent-controlled Dokuchaevsk town, adding that all the injured people had been taken to local hospitals for treatment.
The press service of the Ukrainian army accused independence-seeking insurgents of being responsible for the attack, saying they "intentionally opened fire against civilians."
Some earlier reports put the death toll at six, and quoted regional officials as saying that the bus was hit by a rocket that appeared to be targeted at a road block nearby but have gone astray.
Meanwhile, local media quoted some insurgent commanders as saying that they were not involved.
The attack was one of the worst outbreaks of violence against civilians in recent weeks in a conflict between government troops and pro-independence rebels.
The violent confrontation, which has been raging in eastern Ukraine since mid-April, has left more than 4,700 people dead and over 10,000 wounded.
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