Methotrexate (MTX) is a chemotherapeutic and immunosuppressive drug that can impair spermatogenesis and lead to male infertility. This study examined the effects of different MTX concentrations on TM4 Sertoli cells and evaluated the protective role of quercetin (QT), a natural antioxidant, against MTX-induced oxidative stress.TM4 cells were treated with MTX at the following concentrations: 1 µg/mL /mL, 5 µg/mL /mL, and 10 µg/mL /mL for 24 hours, 48 hours, and 72 hours. Our results show that an IC₅₀ value of MTX in these cells was 7 µg/mL. Quercetin was then applied by itself to the cultures at the following concentrations: 10 µg/mL /mL, 25 µg/mL /mL, and 50 µg/mL /mL, and in combination with MTX at its IC₅₀ concentration, for 24 hours. MTX significantly reduced TM4 cell viability, whereas quercetin treatment markedly alleviated this cytotoxic effect. Levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were significantly higher in MTX-treated cells compared with controls (p < 0.001 and p < 0.01, respectively). In contrast, MDA and ROS levels were significantly decreased in quercetin-treated cells (p < 0.05 and p < 0.01), further supporting its antioxidant activity. In addition, cotreatment with quercetin significantly restored the total antioxidant capacity suppressed by MTX (p < 0.01). These findings indicate that quercetin effectively protects TM4 Sertoli cells against MTX-induced oxidative damage.