Cannabidiol (CBD), a major cannabinoid found in Cannabis sativa L., has been used in the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex. Recently, concerns have been raised regarding the male reproductive toxicity of CBD in animal models, such as monkeys, rats, and mice. In our previous studies, we reported that CBD inhibited cell proliferation in both primary human Sertoli cells and mouse Sertoli TM4 cells. Transcriptomic analysis revealed that in primary human Sertoli cells CBD disrupted DNA replication, cell cycle, and DNA repair, ultimately causing cellular senescence. In this study, we further investigated the molecular changes induced by CBD in mouse Sertoli TM4 cells using RNA-sequencing analyses and compared the transcriptomic profile with that of primary human Sertoli cells. Our findings demonstrated that, unlike in primary human Sertoli cells, CBD did not induce cellular senescence but caused apoptosis in mouse Sertoli TM4 cells. Through transcriptomic data analysis in mouse Sertoli TM4 cells, immune and cellular stress responses were identified. Moreover, transcriptomic comparisons revealed major differences in molecular changes induced by CBD between mouse Sertoli TM4 and primary human Sertoli cells. This suggests that primary human Sertoli cells and mouse Sertoli cells may respond differently to CBD.