The present study was conducted to evaluate the impact of α-Cypermethrin (αCYP), the second most commonly used pesticide in India, on the ovine ovarian granulosa cells (GCs) viability, growth, apoptosis, and steroidogenesis. GCs collected from abattoir-derived ovine ovaries were cultured for 3/6 days in the presence of various concentrations of αCYP (0, 1, 10, 25, 50, and 100 μM). The results revealed a binary effect on GCs, where metabolic activity and viability rates were significantly (p <
0.05) lower from 25 μM onwards. Estrogen concentration was significantly low from the 1 μM dose, whereas progesterone concentration showed a significant increase (10 μM) in the spent media of cultured GCs. The cytotoxicity in the GCs exposed to αCYP revealed significant changes in LDH, ROS, CUPRAC, and GST activity (all at 25 μM) and MDA (at 10 μM) compared to those observed in the control group. The gene expression profiles of cultured GCs showed a significant up-regulation of CYP11A1, FSHR (all at 1 μM), StAR, BAX, and CASP3 (all at 10 μM), 3βHSD1 (at 25 μM), and significant down-regulation of CYP17A1 and ERS2 (all at 25 μM), CYP19A1 and 17βHSD (all at 1 μM), ESR1 and BCL2 (all at 10 μM) in comparison to those observed in control groups. The results of the present experiment demonstrated that αCYP affected the growth and functional parameters of GCs, the expression of steroid hormone-associated genes, and hormone secretion.