Abstract: We explored regional differences in the chemical composition and climate response mechanism for the accumulation of active constituents in Codonopsis pilosula (Franch.) Nannf. based on chemical components and multi-scale climatic factors in Gansu Province. High-performance liquid chromatography (HPLC) was applied to determine the concentrations and differences in the contents of active components in C. pilosula from different regions. Correlation analysis was performed to assess the relationships among annual, quarterly, and monthly climatic factors and chemical components. Results showed regional differences in the chemical component contents in C. pilosula, with atractylenolide Ⅲ content showing the most significant difference across regions. There was no significant correlation between climatic factors and contents of lobetyolin or ethanol-soluble extractives. However, polysaccharide content was positively correlated with mean annual temperature and atractylenolide Ⅲ content was positively correlated with annual precipitation and annual average relative humidity, but negatively correlated with annual average solar radiation. Monthly mean precipitation and monthly mean solar radiation were key factors related to atractylenolide Ⅲ content in C. pilosula. Atractylenolide Ⅲ content was strongly positively related to monthly mean precipitation from March to October, but negatively correlated with monthly mean solar radiation from March to May and July. Precipitation and solar radiation were important climatic factors related to the regional differences in atractylenolide Ⅲ content. High precipitation and low solar radiation were conducive to atractylenolide Ⅲ accumulation in C. pilosula.