The present study was designed to investigate testicular effects of Acyclovir [9-(2-hydroxyethoxymethyl)-9H−guanine] in mouse. Swiss albino male mice (N=6/group/dose/sample time) were treated (i. p.) every day with 4, 16, 32, and 48 mg/kg body weight of Acyclovir for 15 d. The testis was examined for histopathological changes and the seminiferous tubular diameter (STD) and epithelial height (SE) were measured. The sperm count, sperm motility and sperm morphology assay in caudae epididymes, and intra-testicular levels lactate dehydrogenase (LDH) and testosterone were estimated on 7 d, 14 d, 21 d, 28 d, 35 d, and 70 d, following the last exposure. Acyclovir did not affect the body weight, but decreased the testis weight on 21 d and 28 d at two higher dose-levels, and on 35 d at all dose-levels. The STD was decreased on 21 d at 16-48 mg/kg dose-levels (p<0.01), on 28 d and 35 d, at all dose-levels (p<0.01-0.001). The SE was decreased on 14 d at two higher dose-levels (p<0.01), thereafter up to 35 d at all dose-levels (p<0.001). Acyclovir decreased the sperm count from 7-35 d (p<0.001) with a recovery observed on 70 d, except at 48 mg/kg dose-level (p<0.05), and inhibited the sperm motility (p<0.001) from 7-35 d with a maximum effect on 28-35 d. On the other hand, sperm abnormalities increased on 21 d, 28 d and 35 d at 16-48 mg/kg, at 32-48 mg/kg, and at 32 mg/kg dose-levels, respectively (P<0.05). LDH activity was increased (p<0.05-0.001) from 7 d (except at 4 mg/kg) to 35 d. Only 48 mg/kg dose group showed increase in LDH concentration on 70 d. In conclusion, Acyclovir is cytotoxic to germ cells inducing the cellular destruction, and reducing the sperm count and motility, and increasing sperm abnormalities. Further, Acyclovir also caused cellular destruction thus releasing LDH from the cells, and affecting the Leydig cell function. All adverse effects of Acycovir are reversible by 70 d, except the sperm count and LDH level, which appear to be affected over an extended period of time at a higher dose-level.