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Vitamin K2 (VK2) is one of the two types of vitamin K present most in the human diet. VK2 seems to have a beneficial effect on inflammation related to type 2 diabetes mellitus. The aim of this study was to evaluate the influence of VK2 on lipid precursors of inflammation in lipid-overloaded human liver hepatocellular carcinoma cells. Cells were incubated with VK2 and/or palmitic acid (PA). The concentrations of lipid fractions and their fatty acid compositions were measured by gas-liquid chromatography. The expression of proteins involved in the inflammatory process was detected using western blotting. The concentration of triacylglycerols (TAGs), activities of the n-3 pathway in TAGs, and lipooxygenase 15 expression were significantly elevated in cells incubated with PA and VK2. In the same group, a marked elevation in diacylglycerol (DAG) 20:4 was observed. VK2 supplementation lowered the expression of tumour necrosis factor-alpha and interleukin-6 compared to that in the PA group. The data indicate that VK2 redirects fatty acid metabolism into the deposition of a safe TAG fraction by increasing the concentration of anti-inflammatory n-3 polyunsaturated fatty acids in this fraction. Moreover, VK2 stimulates the synthesis of anti-inflammatory factors and has anti-inflammatory effects by reducing DAG 20:4.