Several studies have shown that 1,3-dinitrobenzene (1,3-DNB) causes injury to Sertoli cells and induces apoptosis in the surrounding germinal cells in male laboratory rats; however, the mechanism by which 1,3-DNB functions is not well understood. In this study, we investigated whether 1,3-DNB induces apoptosis and which pathways are undertaken in TM4 cells. When cells were treated with 1,3-DNB, a dose-dependent reduction in cell viability was observed by tetrazolium dye assay and LDH assay. The reduced cell viability by 1,3-DNB treatment appeared to involve necrosis as well as apoptosis, based on staining with annexin V-FITC and propidium iodide (PI) staining and Western blotting for PARP protein. 1,3-DNB treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and increased expression levels of the pro-apoptotic protein Bax. In addition, using FACS analysis we detected G2/M phase cell cycle arrest by 1,3-DNB, concurrent with a remarkable increase in p21 expression and decrease in cdc2 expression. Interestingly, we found that the phosphorylation of c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) was promoted by 1,3-DNB, furthermore, 1,3-DNB-induced cell death was significantly inhibited by the JNK inhibitor, but not by ERK inhibitor or the p38 inhibitor. Together, our results suggest that 1,3-DNB induces apoptotic cell death and G2/M phase cell cycle arrest, at least in part, via JNK signaling in TM4 mouse Sertoli cells.