This study investigated the storage temperature effects on Polygonati Rhizoma probiotics (PRP) activity by establishing storage conditions at different temperatures and regularly detecting the viable bacteria counts and value of pH. To further analyze the metabolic changes, the metabonomics analysis was carried out by using UHPLC-QE-MS/MS. Multivariate analysis used principal component analysis and orthogonal partial least squares discriminant analysis. When the storage temperatures were 25 °C and 37 °C, 229 and 307 different metabolites were identified respectively. The levels of most compounds such as glutamine, asparagine, and citrate decreased. This change affected both the flavor and nutritional benefits of PRP. In addition, the count of viable bacteria at 20 °C and 4 °C is very high, and there is no significant difference. Therefore, considering the product quality and energy saving, we recommend 4 °C as the storage temperature. This research provides important information on the storage stability and shelf-life setting of PRP products.