Abstract: In order to investigate process of supercritical carbon dioxide fluid extraction(SFE-CO₂)of cucurbitacin from Cucumismelo L. stem systematically and comprehensively,the main control variables were comprehensively analyzed. The effects of entrainer amount,extraction temperature,extraction pressure,extraction time,CO₂ flow rate and particle size on the extraction yield of cucurbitacin were studied by single factor experiment. According to the date from the single factor experiments,the orthogonal test was used to optimize the extraction process of cucurbitacin by SFE-CO₂.The best condition for SFE-CO₂ extraction of cucurbitacin was as follows:The entrainer amount of 200 mL,extraction temperature 35℃,extraction pressure 25 MPa,extraction time 30 min,CO₂ flow rate 20 kg·h^-1,particle size 60~80 mesh. In this condition,the extraction yield of cucurbitacin was 1.443%. The extracts were analyzed by HPLC,and the results showed that cucurbitacin B was mainly composed,which contained cucurbitacin B,D,Eand I,the contents were 7.83,1.03,3.25,2.38 mg·g^-1. Results showed that the SFE-CO₂extraction of cucurbitacin could avoid the loss of heat-sensitive cucurbitacin.