Introduction Chronic exposure to pesticides is increasingly becoming a concern in the modern world. Atrazine (ATZ), a widely used pesticide in the U.S., is known to interfere with the male reproductive system. In the present study, we examined the effects of ATZ on viability, cell cycle progression, steroid production, and steroidogenic gene expression profile of testicular Leydig cells using the mouse Leydig cell line I-10 as an in vitro model. We then determined whether curcumin (Cur), an herbal medicine, can counteract the effects of ATZ. Molecular docking was performed to assess the potential of direct binding of ATZ and Cur to key steroidogenic factors of both human and mouse origins. Methods I-10 cells were treated for 6 days with either ATZ (3.3, 10, 33, 100 µM), Cur (3, 10, 30 µM), or a co-treatment of Cur (3 µM) + ATZ (33 or 100 µM). Cell viability, cell cycle progression, production of progesterone (P4) and testosterone (T), and expression of key steroidogenic genes were assessed on Days 2, 4, and 6. Additionally, AutoDock Vina v1.2.7 was used to predict the binding affinities of Cur and ATZ to key mouse and human steroidogenic proteins in comparison to their endogenous substrates. Results ATZ decreased I-10 cell viability while increasing T levels in a concentration-dependent manner. Cur transiently stimulated I-10 cell proliferation and increased P4 but showed mixed effects on T levels. Co-treatment decreased viability, as supported by changes in cell cycle distributions. Co-treatment also significantly increased both P4 and T production, with induction of expression of the steroidogenic genes StAR, Cyp11a1, Hsd3β6, and Hsd17β3 at specific time points. Furthermore, Cur exhibited strong predicted binding to both mouse and human steroidogenic proteins, comparable to that of their endogenous substrates. Conclusion Our findings suggest a potential interaction between Cur and ATZ, which was more prominent at ATZ 100 μM, stimulating steroidogenesis in I-10 Leydig cells at the expense of decreased viability.