The present study aims to evaluate the effects of repeated exposure to 2-ethylhexyldiphenyl phosphate (EHDPP) on human liver cells. In vitro threedimensional (3D) hepatospheroid cell culture was utilized to explore the potential mechanisms of EHDPP-mediated metabolic disruption through morphological, transcriptional, and biochemical assays. Lipidomics analysis was performed on the individual hepatospheroids to investigate the effects on intracellular lipid profiles, followed by hepatospheroid morphology, growth, functional parameters, and cytotoxicity evaluation. The possible mechanisms were delineated using the genelevel analysis by assessing the expression of key genes encoding for hepatic lipid metabolism. We revealed that exposure to EHDPP at 1 and 10 mu M for 7 days alters the lipid profile of human 3D hepatospheroids. Dysregulation in several lipid classes, including sterol lipids (cholesterol esters), sphingolipids (dihydroceramide, hexosylceramide, ceramide, sphingomyelin), glycerolipids (triglycerides), glycerophospholipids, and fatty acyls, was noted along with alteration in genes including ACAT1, ACAT2, CYP27A1, ABCA1, GPAT2, PNPLA2, PGC1 alpha, and Nrf2. Our study brings a novel insight into the metabolic disrupting effects of EHDPP and demonstrates the utility of hepatospheroids as an in vitro cell culture model complemented with omics technology (e.g., lipidomics) for mechanistic toxicity studies.