【Objective】The present study aimed to determine the protective role of Melatonin（MT）against Acrylamide（AA）- induced testicular toxicity and the potential molecular mechanism.【Methods】The animals were randomly divided into three groups， the control group（n = 12），the AA group（n = 12）and the AA+MT group（n = 12）. The rats in the AA and AA + MT group were gavaged with AA at a dose of 15 mg/ （kg·day）for 4 consecutive weeks. After 3 weeks of AA treatment，MT was intraperitoneally injected 30 minutes before AA treatment at 10 mg/ （kg·day）for 1 week in the AA + MT rats. Subsequently，the mitochondrial membrane potential measurement，TUNEL assay，Western blot and electron microscopic techniques were applied in the present study.【Results】The results showed that AA could decrease the testis mitochondrial membrane potential（P < 0.05）which could be recovered by MT（P < 0.05）. Moreover，MT induced down-regulation of Bax expression and up-regulation of Bcl-2 expression in the testis，compared with AA rats. The amelioration of testicular apoptosis was further confirmed by the TUNEL labeling. Western blot results suggested that the decreased ratios of Bcl-2/Bax and Bcl-xL/Bak in the AA group（both P < 0.05）could be recovered by MT treatment（both P < 0.05）. The levels of Cyt-c，Casp-3，p53 and NF-κB in AA group were markedly elevated compared with the control（all P < 0.05），and reduced in MT treatment group（all P < 0.05）. MT could relieve abnormal mitochondrial structures in the seminiferous tubule in the electron microscopic level.【Conclusion】MT may exert productive effect through its anti-apoptotic properties associated with mitochondria.