Abstract: BACKGROUND: Supercritical fluid extraction with CO2 (SFE‐CO2) was utilized for extraction of capsaicin (CA) and dihydrocapsaicin (DHCA) from Capsici Fructus, and then a two‐step enrichment method for separating capsaicinoids from SFE‐CO2 extracts was developed. The process involved extraction with aqueous methanol and crystallization by alkali extraction and acid precipitation. Finally, a consecutive high‐speed countercurrent chromatography (HSCCC) separation method was successfully applied in the purification of CA and DHCA from capsaicinoid crystal. RESULTS: The extraction pressure, extraction temperature and volume of co‐solvent were optimized at 33 MPa, 41 °C and 75 mL, respectively, using response surface methodology; the extraction rates of CA and DHCA were about 93.18% and 93.49%, respectively. 407.43 mg capsaicinoid crystal was isolated from the SFE‐CO2 extracts obtained from 100 g capsicum powder by the two‐step enrichment method. About 506 mg and 184 mg CA and DHCA with purities up to 98.31% and 96.68%, respectively, were obtained from 1 g capsaicinoid crystal in one HSCCC of three consecutive sample loadings without exchanging any solvent system. CONCLUSIONS: This method comprising SFE‐CO2, a two‐step enrichment and HSCCC was efficient, powerful and practical for the large‐scale preparation of CA and DHCA from Capsici Fructus with high purity and high yield. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]