In recent years, with the rapid increase of smoking in the world, male reproductive toxicity induced by cigarette smoking (CS) has attracted increasing attention. Studies have shown that long-term heavy smoking can lead to testicular damage in men, resulting in decreased semen quality, but the specific mechanism is still unknown. This study aimed to investigate the regulation mechanism of the Bcl-2 signaling pathway in rat testicular apoptosis induced by CS exposure. SPF male SD rats were randomly divided into high, medium and low CS exposure groups (30, 20 and 10 non-filtered cigarettes/ day, respectively) and control groups. The rats in each group were treated with static exposure methods and were anesthetized after 2, 4, 6, 8 and 12 weeks-CS exposure, respectively. The testicular organ coefficient was calculated, and the testicular histopathological changes and apoptosis in rats were detected. The mRNA and protein expressions of Apaf-1, Caspase-9, Bim, Bcl-w and Bak in the mitochondrial apoptosis pathway were detected. Results indicated that with the increase of exposure dose and duration, the weight of rats in exposure groups gradually decreased, the testis in exposure groups showed obvious pathological changes, and the testicular organ coefficient gradually decreased. Compared with the control groups, the testicular organ coefficient in the middle, high-dose groups at the 8th week and all groups at the 12th week significantly decreased (P < 0. 05). The number of positive apoptotic cells in the testis increased with the prolongation of exposure time, and it showed statistical difference since six weeks (P < 0. 05). After 12 weeks of CS exposure, the mRNA transcription and protein expression of Apaf-1 and Caspase-9 in the rat testis were significantly up-regulated compared with control groups (P < 0. 05). The mRNA expression level of the pro-apoptotic gene Bak gradually decreased in the early stage of exposure, and increased since the eighth week of exposure; the protein expression of Bak was up-regulated, and mRNA transcription and protein expression of Bak were significantly up-regulated at 12-week exposure groups compared with control groups (P<0. 05). The mRNA expression of the anti-apoptotic gene Bcl-w was significantly down-regulated since the 6th week of exposure (P < 0. 05), the protein of Bcl-w was significantly down-regulated since the 12th week of exposure (P < 0. 05). The mRNA and protein expression of BH3-only gene Bim was up-regulated, but there was no statistical difference compared with control groups (P > 0. 05). In conclusion, long-term heavy smoking activated the mitochondrial apoptosis pathway and induced testicular irreversible apoptotic damage. These results provide a scientific basis for further study of the molecular mechanism of male reproductive injury induced by cigarette smoking.