Natural sources of vitamin D (VD), have been proposed to represent an alternative to synthetic vitamin D in nutritional interventions, also holding therapeutic potential in non-alcoholic fatty liver disease (NAFLD). In this study lipidomics was used to comparatively investigate the molecular mechanisms behind the therapeutic effects of a natural VD formulation consisting of a Shitake mushroom extracts (NVD) and a synthetic cholecalciferol formulation (SVD) in HepaRG human hepatocytes exposed to free fatty acid (FFA)-induced lipotoxicity. The results demonstrate that the two VD formulations prevent lipotoxicity with similar efficacy, but different lipidomic fingerprints. Differentially expressed lipids in NVD’ in vitro therapeutic effect indicated a reduced synthesis of cellular triglycerides; combined with a marked reshaping of glycerophospholipid metabolism and characteristic changes of the chain length and number of double bonds in the phosphatidylcholine pool that were absent in SVD treatment. Bioinformatics interpretation of lipidomics data associated NVD therapeutic properties to an enhanced insulin function and glycerophospholipid metabolism, whereas SVD was primarily associated with the inflammatory signaling and death pathways of the liver cell. These differences between the two VD formulations were further highlighted matching lipidomics data with gene microarray (transcriptomics) data available from previous studies on this experimental model; the resulting multiomics data identified lipid metabolism nodes specific for the multimolecular mechanisms of the two formulations which may deserve further pre-clinical investigation in the treatment of hepatocyte lipotoxicity.