Internal reproductive organ size is an important determinant of male reproductive success. While the response of testis length to variation in the intensity of sperm competition is well documented across many taxa, few studies address the importance of testis size in determining other components of male reproductive success (such as mating frequency) or the significance of size variation in accessory reproductive organs. Accessory gland length, but not testis length, is both phenotypically and genetically correlated with male mating frequency in the stalk-eyed fly Cyrtodiopsis dalmanni. Here we directly manipulate male mating status to investigate the effect of copulation on the size of both the testes and the accessory glands of C. dalmanni. Accessory gland length was positively correlated with male mating frequency. Copulation induced a significant decrease in accessory gland size. The size of the accessory glands then recovered slowly over the next 8–48 hours. Neither testis length nor testis area was altered by copulation. These results reveal that the time course of accessory gland recovery corresponds to field observations of mating behaviour and suggest that accessory gland size may limit male mating frequency in C. dalmanni.