1. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOF MS)-based lipidomics was employ to elucidate new mechanism of alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis in mice. 2. Multiple lipid components significantly increased in ANIT-induced intrahepatic cholestasis, including PC 16:0, 20:4, PC 16:0, 22:6, PC 16:0, 18:2, LPC 18:2, PC 18:2, LPC 18:1, PC 18:1, 14:0, SM 18:1, 16:0, oleoylcarnitine, and palmitoylcarnitine. This alteration of lipid profile was induced by the changed expression of genes choline kinase (Chk) a, sphingomyelin phosphodiesterase (SMPD) and stearoyl-coenzyme A desaturase 1 (SCD1). 3. Knockout of Aryl hydrocarbon receptor (Ahr) in mice can significantly reverse alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis, as indicated by lowered ALT, AST and ALP activity, and liver histology. Aryl hydrocarbon receptor (Ahr) knockout significantly reversed alpha-naphthyl isothiocyanate (ANIT)-induced lipid metabolism alteration through regulating the expression of Chka. 4. In conclusion, this study demonstrated ANIT-induced lipid metabolism disruption might be the potential pathogenesis of ANIT-induced intrahepatic cholestasis in mice.