Purpose: Hirsutism (ie, terminal hair growth on the face and body in a male-like pattern in women) is a common dermatological disorder in women, with psychosocial implications. Consequently, there is demand for finding novel pharmacological treatments and agents that can safely reduce hair growth. This study aimed to investigate the potential effect of topical acyclovir on hair growth in mice. Methods: In this study, twenty-four female BALB/c mice were randomly divided into three groups in order to evaluate the hair growth-reducing effects of acyclovir (control group, vehicle group, and acyclovir group). Topical acyclovir 5% was applied on the shaved denuded skin of mice. Topical application onto the backs of the animals was performed twice daily for 28 consecutive days. The time (in days) required for hair growth initiation as well as completion of hair growth in dorsal skin of animals were recorded. On day 28, horizontally cut biopsy samples were removed and the numbers of hair follicles were counted, and the diameter of hair follicles was measured under high-field microscopy by a specialist blinded to the treatments. Results: Hair growth initiation time was significantly increased with acyclovir, as compared to control and vehicle groups. The time required for complete hair growth in control and vehicle groups were 18±0.68 and 19±1.41 days, respectively; however, the hair growth completion in acyclovir-treated animals was not observed at the end of the experiment. Furthermore, the length of hairs in treatment group was significantly shorter than the control group at the end of the study (P < 0.001). In histologic examination, the count and the diameter of hair follicles in deep subcutis were significantly decreased. Conclusion: The results of this study, for the first time, showed that topical administration of acyclovir might have inhibitory effects on hair growth in experimental animals; however, further studies are required to understand its mechanism.