Two people died on A436 near Pegglesworth at 11:15 GMT on Monday after the crash between two vehicles.
The crash took place near Pegglesworth, between the A435 (Seven Springs) and the Kilkenny Inn, when a Mazda had been traveling towards Seven Springs and the HGV had been heading in the opposite direction.
Both vehicles crashed with each other.
The lorry driver had minor injuries.
Three of the Mazda’s passengers were admitted to Bristol’s Southmead Hospital and John Radcliffe Hospital in Oxford.
A 24-year-old woman who had been traveling in the car succumbed to her injuries in hospital on Monday night.
The driver of the car, a 31-year-old man, was pronounced dead at the scene.
A one-year-old baby and a 29-year-old woman are in serious condition.
A 24-year-old man had received treatment and was discharged.
Road closures were conducted but they were later removed.
Gloucestershire Constabulary has urged witnesses to contact them about the crash.
