In order to further explore the by-products of Gynostemma pentaphyllum (Thunb.) Makino, the seed oil was studied for its extraction, physicochemical properties and bioactivity. Supercritical carbon dioxide (SC -CO2) extraction was the first time to extract G. pentaphyllum seed oil (GPSO). The maximum extraction yield of 35.96 +/- 0.21% was achieved at the optimal conditions: extraction temperature of 43 degrees C, extraction pressure of 32 MPa and extraction time of 160 min, which were selected and optimized through response surface methodology. The oil yield and physicochemical properties of GPSO obtained by different extraction methods were analyzed and compared. Fatty acid composition was determined by gas chromatography-mass spectrometry and the structure of seed powder was observed using scanning electron microscope. The results showed that SC-CO2 extraction was an effective method to improve GPSO quality and tended to extract higher percentage of unsaturated fatty acids (95.69%) especially conjugated linolenic acid (88.17%) in comparison with conventional extraction methods. Furthermore, the cytotoxicity of fatty acids from GPSO (GPFA) was investigated by cell viability assay and cell morphological changes. GPFA significantly inhibited cell viability in a concentration-dependent manner against various cancer cells especially leukemia cells, suggesting its potential benefits as an anticancer agent. (C) 2016 Elsevier B.V. All rights reserved.