<p>Overripening of ovulated oocytes, often resulting from spawning delays in aquaculture, is triggered by apoptosis and significantly compromises early fertilization in fish. Targeting apoptosis pathways offers a promising approach to enhance reproductive outcomes, particularly given the controversial role of melatonin in apoptosis regulation, this study assessed the impact of oral melatonin supplementation at doses of 0, 50, and 200 mg/kg of dry feed on the expression of key apoptosis-related genes (Bcl-2, p53, caspase-9) and fertilization percentages in rainbow trout (<i>Oncorhynchus mykiss</i>) oocytes at two post-ovulation time points: immediately post-ovulation (day 0) and after 7 days of post-ovulatory ageing (overripening). Gene expression was quantified using real-time PCR, and fertilization percentages were evaluated to determine reproductive outcomes. Melatonin treatment notably upregulated the anti-apoptotic gene Bcl-2 and downregulated the pro-apoptotic genes p53 and caspase-9. While no significant differences were detected between the 50 and 200 mg doses for p53 and caspase-9 expression, the 200 mg dose demonstrated a more pronounced effect on improving fertilization percentages in overripened oocytes. In contrast, fertilization percentages declined sharply in the control group during overripening. Both melatonin dosages significantly improved fertilization percentages compared to the control, with higher doses showing greater effectiveness. These results suggest that melatonin alleviates the detrimental effects of oocyte overripening by modulating apoptosis pathways and enhancing fertilization outcomes. Melatonin supplementation may be a practical approach in aquaculture to extend oocyte viability and improve reproductive performance in delayed spawning scenarios.</p>