Abstract: Using Biolog–Eco technology, the bacterial community composition and diversity in the soil of different soil depths(0−20, 20−40 cm and 40−60 cm) of Paeonia ostii cultivation were studied, the vertical distribution of bacterial carbon source metabolism ability and carbon source utilization mode in different soil layers were explored. The findings indicated that with the increase of soil depth, the average color change rate(AWCD) of P. ostii rhizosphere soil bacterial community showed a significant downward trend; Shannon index and Simpson index also showed a significant downward trend with the increase of different soil depths; the carbon source metabolic capacity of the bacterial community decreased significantly with the increase of different soil depths; carbohydrates, amino acids, carboxylic acids and amines were the main metabolic methods of the rhizosphere bacterial community; the rhizosphere soil bacteria at different soil depths were mainly affected by pH, SOC, TN and C/N. Therefore, the carbon source metabolism and functional diversity of soil bacteria in P. ostii at different depths have changed, and the types of carbon sources used by bacteria at different soil depths are also different. The physical and chemical properties of soil are responsible for the changes in bacterial functions at different soil depths.