This study aims to determine the effects of Rhodobacter sphaeroides 8513 on the physiological and biochemical indicators, short-chain fatty acids, and gut microbiota of healthy BALB/c mice. Mice were divided into the control group and R. sphaeroides group (5×10 8 CFU/mL). After 28 days of continuous gavage, fecal samples were collected for high-throughput sequencing, then mice were sacrificed and the serum was taken for biochemical analysis. Liver, kidney, and spleen were obtained for organ coefficient calculation, and cecum contents were collected for short-chain fatty acid analysis by gas chromatography. The results showed that there was no significant difference in body weight gain, alanine aminotransferase (ALT) and urea nitrogen (BUN) levels between the control group and R. sphaeroides group, while R. sphaeroides significantly decreased the aspartate aminotransferase (AST) and creatinine (CRE) level. No side effect of R. sphaeroides treatment on the basic physiological health of mice was observed. R. sphaeroides significantly increased the content of acetic acids in the gut of mice, while there were no significant changes in the concentration of butyric and propionic acid. High-throughput sequencing analysis indicated that there was a significant difference in α-diversity between these 2 groups, while there was no significant difference in β-diversity between them. R. sphaeroides not only increased the abundance of anaerobic bacteria of Rhodospirillaceae, Desulfovibrionaceae, and Helicobacter in the gut of mice, but also increased the abundance of the predominant microflora in the gut such as Bacteroidaceae (Bacteroides), Prevotellaceae, Clostridiales_vadinBB60_group (Closporaceae). These results suggested that R. sphaeroides 8513 could modulate the microbial ecology in the gut of mice.