They were being transported by lorry to Birmingham when they were involved in a one vehicle road traffic collision on the A76 at Enterkinfoot near Sanquhar.
It happened at about 10.50 pm on Monday night and resulted in the closure of the road overnight until 5 am on Tuesday.
The driver of the Scania HGV, a 27-year-old man from Carlisle, suffered minor injuries but did not require hospital treatment.
However, of the 440 sheep on board, 220 of them died in the crash or were subsequently put down by local vets.
Due to the nature of the collision, staff from Police Scotland, Scottish Environmental Protection Agency, Scottish Fire and Rescue Service, Scottish Ambulance Service and local farmers and vets were all involved in the incident.
Anyone who witnessed the incident should contact Police Scotland on 101, quoting incident 4406.
