Background <p>In vitro semen preservation is a crucial technique for conserving genetic material from male dogs. However, during the storage, oxidative damage can compromise key sperm structures, resulting in the decline of sperm viability, affecting their recognition of oocytes, and even causing sperm death. Therefore, the addition of exogenous antioxidants to alleviate the oxidative damage to sperm has become an important strategy to improve the effect of sperm storage in vitro. <i>Astragalus polysaccharide</i> (APS) is a kind of water soluble heteropolysaccharide with high-efficiency antioxidant activity, but its effect and mechanism in the preservation of canine semen remain to be studied. Thus, the present was designed to investigate the effects of APS on canine sperm after chilled storage, as well as its mechanism.</p> Results <p>It was observed that the supplementation of 0.50&#xa0;mg/mL APS significantly improved sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, enhanced sperm antioxidant capacity, and promoted AMPK phosphorylation, while reduced the level of lipid peroxidation, after chilled storage. In addition, the effects of APS were partly counteracted when oxidative stressed sperm induced by H₂O₂ was incubated with 0.50&#xa0;mg/mL APS and 40 µM Compound C (an AMPK inhibitor). These results suggest that APS protects canine sperm after chilled storage via promoting AMPK phosphorylation.</p> Conclusion <p>The supplementation of APS to the extender protects canine sperm after chilled storage via promoting AMPK phosphorylation for enhancing the antioxidant capacity of sperm. These findings provide novel insight into the mechanism of APS in protecting canine sperm after chilled storage, and reveal the feasibility of APS supplementation to sperm extender for canine sperm preservation.</p>