This study aimed to investigate the effects of gossypol on cell viability, oxidative stress, and gene expression in Primary Smooth Muscle of Myometrium from Ovis aries (PSMo24) Cells. The cells were cultured and treated with varying concentrations of gossypol (1-2000 µg/mL), cytotoxicity was assessed using the MTT assay, oxidative stress were evaluated by fluorescence-based methods (DCFH-DA/ABAP), and the expression of structural, hormonal, apoptotic, and antioxidant genes was analyzed by RT-qPCR. The results showed that concentrations ≤ 5 µg/mL gossypol (G5) maintained cell viability, whereas higher doses induced a progressive loss of viability, indicating dose-dependent cytotoxicity. The production of reactive oxygen species was not significantly affected, suggesting that gossypol did not exhibit relevant antioxidant or pro-oxidant activity at the tested doses. Gene expression analysis revealed differential regulation of β-actin, myosin light chain kinase (MLCK), progesterone receptor (PR), estradiol receptor (ER), the pro-apoptotic gene BAX, and the anti-apoptotic gene BCL2. The transition observed between G7, G10, and G15 revealed a functional gradient: G7 reflected a cell survival profile; G10 marked the apoptotic inflection point; and G15 corresponded to programmed cell death. The antioxidant genes remained unchanged, indicating the maintenance of cellular oxidative homeostasis. In conclusion, gossypol induces dose-dependent cytotoxicity in uterine smooth muscle cells (PSMo24), modulating structural, hormonal, and apoptotic genes without altering basal antioxidant activity. Low concentrations were well tolerated, while higher doses from 7 µg/mL activated pro-apoptotic pathways The results highlight the need for caution in reproductive contexts within the animal field and suggest potential use in oncological therapies.