The accident occured on the B725 between the villages of Clarencefield and Bankend, near Dumfries, at about 5.15pm tonight.
Police Scotland Dumfries and Galloway division said that a vehicle and two horses and riders were caught up in the crash.
The force said the woman, who came from the Clarencefield area, died. Another local female, who is also thought to have been riding a horse, is being treated for serious injuries.
Police Scotland are appealing for any persons who witnessed the accident, or were in the area around that time and have any information to telephone police on 101
Motorists have been warned the B725 Glencaple to Clarencefield Road has been closed, following a Road Traffic Collision near to Locharwood, Clarencefield.
Police said the road will be closed for some time and diversions are to be put in place and advised people to avoid the area.