Updated FTP location. Monoethylhexyl phthalate (MEHP) is one of the main active metabolites of the plasticizer di(2-ethylhexyl) phthalate (DEHP). It has been known that MEHP has impact on lipolysis; however, its mechanism on the cellular lipid metabolism remains largely unclear. Here, we first utilized the global lipid profiling to fully characterize the lipid synthesis and degradation pathways upon MEHP treatment. Meanwhile, we further identified the possible MEHP targeted proteins in the living cells using the cellular thermal shift assay (CETSA) method. The lipidomics result showed that there was a significant accumulation of fatty acids and other lipids in cell. The CETSA identified 138 proteins and fatty acid β-oxidation inhibition pathways that were significantly perturbed. MEHP’s binding with selected proteins HADH and HSD17B10 was further evaluated using molecule docking and results showed that MEHP has higher affinities as compared to the endogenous substrates, which was further experimentally confirmed in the surface plasma resonance (SPR) interaction assay. In summary, we found a novel mechanism for MEHP-induced lipid accumulation, which was probably due to its inhibitive effects on the enzymes in fatty acid β-oxidation. This mechanism substantiates the public concerns on the high exposure level to plasticizers and its possible role as an obesogen.