To investigate the effect of probiotic Lactobacillus fermentum CKCC1858, LF on the prevention of hyperlipidemia and its correlation with gut microbiota, golden hamsters were fed a high-fat diet alone or in combination with the probiotic for 6 weeks. The results showed that the LF intervention alleviated HFD-induced hyperlipidemia and liver damage, as evidenced by the reduced serum lipid profile levels and liver function markers. More importantly, the LF intervention attenuated HFD-induced microbiota dysbiosis by enhancing the abundance of SCFA-producing bacteria and reshaping the metabolic functions of the gut microbiota, likely contributing to its pronounced preventive effects on hyperlipidemia. This study elucidated the mechanism of the preventive effect of probiotics on hyperlipidemia in terms of regulating gut microbiota, and provided suggestions for regulating gut microbiota through probiotic interventions to improve lipid metabolism.