Abstract: Objective: To investigate the relative content of polyunsaturated fatty acids (PUFAs) in Portulaca oleracea and the effect of Portulaca oleracea purified oil on lipid accumulation in HepG2 cells. Methods: The fatty acid composition of whole herb oil and purified oil were analyzed by gas chromatography-mass spectrometry. MTT method was used to determine the effect of different concentrations of Portulaca oleracea purified oil on cell viability and the suitable concentration range was selected for subsequent experiments. Oil red O staining was used to determine whether the oleic acid-induced lipid accumulation model was successfully established. The kit method was used to determine the effects of purified oil concentration on high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), total cholesterol (TC) and triglyceride (TG) levels in low, medium and high dose groups (60、80、100 μg/mL). Results: There were three kinds of PUFAs in Portulaca oleracea include linoleic acid, α-linolenic acid and γ-linolenic acid. The relative content of PUFAs in the whole herb oil of Portulaca oleracea was 27.6104%, and the relative content of purified oil after enrichment and purification was as high as 73.9015%. The concentration range of purified oil selected by MTT method was 60~100 μg/mL. Compared with the model group, the concentration of HDL-C in the high-dose group (100 μg/mL) of Portulaca oleracea purified oil was significantly increased, and the concentrations of LDL-C, TC and TG were significantly decreased (P<0.01). Conclusion: The relative content of PUFAs in purified oil of Portulaca oleracea was relatively high, which basically achieved the purpose of purification. PUFAs of Portulaca oleracea had good relief effect on fatty liver, showing strong lipid-lowering ability in vitro.