This study is the first to investigate the effects of tebuconazole (TEB) on the physiological functions of bovine testicular cells and epididymal spermatozoa. Motility and plasma membrane integrity of spermatozoa exposed to TEB (0.001-100 µM) were evaluated at different incubation times (0-6 h), while TEB-induced spermiotoxicity was assessed after 24 h in cell cultures. Testicular cells, obtained from the parenchyma of bovine testes, were seeded at 1.0 × 104 and 1.5 × 106 cells/well in 96- and 12-well culture plates and incubated for 48 h in culture media containing TEB (0.001-100 µM) to evaluate cytotoxicity and hormone release, respectively. TEB did not affect the motility and plasma membrane integrity. However, significant spermiotoxicity occurred at higher TEB (1-100 µM) concentrations (P < 0.05) compared to control and lower doses. Although no dose caused cytotoxicity in testicular cells (P > 0.05), 1 and 100 µM TEB caused a significant increase in testosterone secretion (P < 0.05). As a result, high doses of TEB (1-100 µM) had slightly suppressive effects on spermatozoa; however, these doses had stimulatory effects on testosterone secretion by testicular cells. It appears that the disruption of hormonal homeostasis of testicular cells after TEB exposure may result in metabolic and especially reproductive adverse effects in bulls.