Many seasonally breeding mammals use changes in photoperiod as a reliable cue to time reproduction. Photoperiodic timing assists an animal in predicting annual environmental changes in its habitat and therefore, enables it to accurately time reproductive events to the most favourable conditions. Changes in day length are more pronounced in the temperate regions and photoperiod is used as a cue for reproduction by most mammals above 30° latitude; however, a number of subtropical species also use this proximate factor to regulate their reproductive cycle. We investigated the reproductive photoresponsiveness of 14 male spiny mice (Acomys spinosissimus) from southern Africa to short-day (SD; 8 h light : 16 h dark) and long-day (LD; 16 h light : 8 h dark) photoperiods. Testicular mass and volume, seminiferous tubule diameter and plasma testosterone concentrations significantly increased in animals subjected to LD and they were regressed when the males were kept under SD. Body mass of the males was not significantly affected by the photoperiodic conditions. Although male A. spinosissimus appear to use photoperiod as a proximate factor to regulate reproduction seasonally, other environmental factors, such as rainfall, food quantity and quality as well as temperature, may regulate reproduction in A. spinosissimus in concert with photoperiod. In conclusion, the present study demonstrates the significance of photoperiodic time-measuring systems in the regulation of seasonal reproduction in a subtropical rodent.