Stropharia rugosoannulata is abundant in natural polysaccharides, offering not only high nutritional value but also significant medicinal properties, potentially useful in managing hyperlipidemia. This study aimed to assess the hypolipidemic effects of S. rugosoannulata polysaccharide in mice with hyperlipidemia caused by a high-fat diet (HFD). We examined the influence of S. rugosoannulata polysaccharide on protein expression in the LKB1/AMPK pathway using Western blot analysis. The results showed that S. rugosoannulata polysaccharide increased the expressions of LKB1 and AMPK, decreased the expressions of CD36, SREBP1, ACC1, FAS, and HMGCR, and effectively alleviated the accumulation of liver lipids. Furthermore, in HepG2 cells exposed to oleic acid, mirroring lipid accumulation, we elaborated on these animal study findings. Our results demonstrated that weight gain was inhibited, body fat content was decreased, and dyslipidemia and liver steatosis were improved in mice fed with an HFD after treatment with S. rugosoannulata polysaccharide. The findings of this study indicate that S. rugosoannulata polysaccharide can regulate the liver LKB1/AMPK signaling pathway and inhibit lipid accumulation, thus alleviating hyperlipidemia.