Inflammation, especially the release of pro-inflammatory mediators, contributes to hepatocyte injury during cholestasis. Alpha-naphthylisothiocyanate (ANIT) is widely used in rodents to mimic clinical cholestasis. Lymphocytes have been reported to exacerbate ANIT - induced hepatotoxicity. However, which cell and mechanism mediate hepatic inflammatory response and hepatocyte injury in cholestasis is still not clear. Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes which are supposed to exert immune-regulatory effect on cholestatic liver damage. In the present study, we hypothesized that iNKT cells played a role in the pathogenesis of ANIT-induced cholestatic hepatotoxicity. ANIT (50 mg/kg, intragastric gavage) was administered to male mice for 16, 48, or 72 h. We found that ANIT administration activated iNKT cells, releasing Th1 cytokine IFN-γ and Th2 cytokine IL-4. Administration of ANIT induced cholestatic liver injury, evidenced by the elevated serum ALT, AST, ALP, TBA, TG and TC levels, and significant hepatic histopathological changes. However, knockout of iNKT cell were resistant to the late development of ANIT - induced liver injury due to the reduced release of inflammatory cytokines CXCL10 and ICAM-1, as well as the down-regulation of nuclear receptor Egr1. We further revealed that the improvement of ALP in iNKT cell - deficient mice was partly associated with the up-regulation of transporter MRP2 and NTCP and bile acid metabolism enzyme CYP2B10. Collectively, these results suggested that iNKT cells aggravated ANIT-induced cholestatic liver injury by inducing inflammatory response which contributed to the understanding of the mechanisms of ANIT-induced cholestasis. More importantly, the iNKT cell regulation may promote effective measures that control cholestasis.