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To investigate anticancer effects of Kaempferia parviflora, rhizomes of K. parviflora were extracted with supercritical CO2 fluid and ethanol under various extraction conditions. Antiproliferative activity of these extracts was evaluated against human cervical cancer HeLa and human gastric adenocarcinoma AGS cell lines. Supercritical CO2 fluid extracts (SFEs) of K. parviflora showed more potent antiproliferative activity than the ethanol extracts against both cell lines, because SFEs contained higher concentration of polymethoxyflavones (PMFs) (72–89 wt%) than the ethanol extract (55–70 wt%). From the compositional analysis of the extracts, 5,7-dimethoxyflavone (1) in K. parviflora was proposed as a potent anticancer PMF. Furthermore, it was found that PMFs with more methoxy groups do not necessarily show better anticancer activity.