Ethambutol (EMB) is conventionally used to treat tuberculosis and atypical Mycobacterium infections in combination with other antimycobacterial drugs. Eventually, EMB testicular toxicity has not been explored extensively yet. The aim of the study is to evaluate testicular toxicity of EMB. We explored the impact of EMB on male rats’ fertility, testosterone level and germ cells state, testicular pro- and anti-oxidant status and DNA damage, as well as identified EMB effects on cytochrome P-450 2E1 (CYP2E1) both with computer simulation and in vivo. We demonstrated that EMB administration to male rats decreased in epididymal sperm count (19%) and fertility index (53%). These events were accompanied by reduction in serum testosterone content (1.6 times) and appearance of spermatogenic epithelium damages. It was also found in testes the intensification of lipid peroxidation, decrease in reduced glutathione content and changes in DNA fragmentation. Additionally, computer simulation showed direct interaction of EMB with CYP2E1 active site and heme. On the top of this, we demonstrated that level of testicular CYP2E1 messenger RNA in EMB-treated rats was increased 8.7 folds and p-nitrophenol hydroxylase activity in testes rose three folds. As this shows, EMB-caused CYP2E1 induction, oxidative stress, and apoptosis in the testes contribute to inhibition of steroidogenesis enzymes and spermatogenesis disruption.