The circadian rhythm of locomotor activity in the spiny mouse Acomys spinosissimus from South Africa was investigated under controlled laboratory conditions. Nine individuals were subjected to six successive light cycles of approximately 2 weeks each as follows: (1) a standard light/dark (12:12LD) cycle; (2) a period of constant darkness (DD); (3) a second standard light/dark (12 L:12D) cycle; (4) an inverse of the LD (12:12DL) cycle; (5) a short day cycle (8:16LD); and (6) a long day cycle (16:8LD). All the animals exhibited entrainment of their activity to the LD and DL lighting regimes. Locomotor activity of A. spinosissimus occurred predominantly during the dark phases of the LD, DL, long day and short day cycles. Under LD, the mean percentage of activity was 88.7 ± 0.07% during the dark phase. When subjected to constant darkness, all animals expressed free-running rhythms of locomotor activity (mean ± 1 standard deviation = 23.81 ± 0.33 h; range = 23.2–24.1 h). On the reverse LD cycle, the mean percentage of activity was 81.4 ± 0.09% during the dark phase of the cycle. Mice exhibited significantly more daytime activity during the long day cycle (20.3 ± 5.8%) and no significant change in dark phase activity during the short day cycle (90.1 ± 4.01). The spiny mouse possesses a circadian rhythm of locomotor activity that entrains strongly to light. Locomotory activity occurs predominantly during the dark phase and can therefore be considered a nocturnal mammal.