Assisted reproductive technologies revolutionize animal breeding, notably improving genetics and production efficiency. Among these techniques, semen collection, cooling, and cryopreservation are key for accessing genetically superior stallions. However, preserving stallion sperm poses greater challenges compared to other species. This study aimed to explore the impact of zinc (Zn) sulphate supplementation on the in vitro quality of equine sperm during cold storage. Semen from three stallions was collected and diluted 1:1 (vivo) with the extender INRA96. Sperm morphology was assessed with Diff-Quick®, motility and kinetic parameters performing CASA, as well as membrane integrity and acrosomal reaction using a flow cytometer. Assessments were conducted on fresh samples, at 24- and 48-hour post-cooling, as well as following a heat-resistance test (240 min incubation at 37°C). In experiment 1, four Zn sulfate concentrations were tested: 0 mM, 1 mM, 2 mM, and 3 mM. In experiment 2, a broader range of concentrations of 0 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, and 3.2 mM was assessed alongside the heat-resistance test. Data was analyzed with the statistical package SPSS v15.0. Our findings indicate that incorporating varying Zn concentrations to INRA96 extender doesn't significantly enhance sperm quality of cooled stallion semen assessed at 24 or 48 hours. Additionally, no benefits were observed for any concentration after the heat-resistance test. However, Zn concentrations surpassing 3 mM exhibited detrimental impacts on stallion’s sperm quality parameters. These findings contribute to the understanding of Zn supplementation as a strategy for improving semen preservation in stallions and highlight the importance of maintaining a delicate balance in Zn concentrations to ensure optimal sperm function.