Abstract: Objective: The protective effect of Polygonatum odoratum polysaccharides (POP) on D-galactose (D-gal) induced senescence in rat aortic smooth muscle cells (A7r5) was investigated. Methods: Firstly, the POP was extracted by the water extraction and alcohol precipitation methods. Next, the phenol concentrated sulfuric acid method was applied to further measure the total sugar content of POP. In addition, the A7r5-senescent cell model was established by the D-galactose treatment. Finally, the MTT assay, β-galactosidase staining, reactive oxygen species (ROS) staining as well as JC-1 staining were applied to further detect the protective effect of POP in A7r5-senescent cells induced by D-galactose. Results: This study found an yield of POP was 8.23% and its total sugar content was 76.48%±0.036%. As a result, the MTT assay showed the viability of the A7r5 cells was increased to 174.89%±3.30% after 24 h treatment by 400 μg/mL of POP, while it was decreased to 65.93%±1.63% after 48 h treatment by 40 mg/mL of D-galactose. Interestingly, when a concentration of POP was 100 μg/mL, this study had found the most significant protective effect in A7r5-senescent cells induced by D-galactose, which increased its cell viability to 226.87%±12.58% as compared with the control group (P<0.001). Additionally, POP decreased the total β-galactosidase, inhibited its ROS levels, increased the number of positive cells after JC-1 staining, as well as protected the stability of mitochondrial membrane potential in A7r5 cells induced by D-galactose. Conclusion: POP had a potential role in the suppression of oxidative stress and cellular senescence in A7r5 cells induced by D-galactose, which could further support its anti-aging effects in vitro.