A retrospective study was conducted to investigate the cause of rectal prolapse in working donkeys in Ethiopia. Analysis of data on rectal prolapse cases obtained from the Donkey Health and Welfare Project clinic at the School of Veterinary Medicine, Addis Ababa University, from 1995 to 2004 revealed that 83.6% (n=177) of the cases were associated with Gasterophilus nasalis. The rest 10.7% and 5.7% were associated with work-related (overloading) cause and diarrhoea, respectively. The mean and median numbers of G. nasalis recovered from the rectum of infected donkeys were 66 and 64, respectively, with a range of 2–195. Over 100 G. nasalis larvae were recovered from the rectum of 22% of the donkeys. Circular demarcated ulcer-like and deep circumferential pits or ring-like mucosal lesions were found at the larval attachment sites. G. nasalis infection and the associated rectal prolapse were observed year round. However, the intensity of rectal larval infection and incidence of rectal prolapse were significantly higher during the rainy season (P<0.01). Age and sex of the donkeys had no significant effect on the intensity of rectal larval infection and incidence of rectal prolapse (P>0.05).