Aim To determine the role of Niemann-Pick C1-Like 1 (NPC1L1) in hepatic steatosis induced by liver X receptor (LXR) agonist in mice. Methods After being fed with 0.015% cholesterol diet for 21 days and gavaged with vehicle or T0901317 [25 mg/(kg·day)] for 7 days, both C57BL/6 mice and NPC1L1 knockout (NPC1L1-KO) mice were anaesthetized, livers were weighed and hepatic lipids were extracted and measured by enzymatic methods. Relative hepatic sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) mRNA levels were analyzed by real-time quantitative PCR. Results After being gavaged with T0901317 for one week, the livers of C57BL/6 mice enlarged from 1.1±0.1 g to 2.8±0.3 g, and hepatic triglyceride content markedly increased from 34.2±18.1 mg/g to 232.2±67.9 mg/g, which was associated with the significantly enhanced hepatic mRNA levels of SREBP-1,FAS and SCD-1. However in NPC1L1-KO mice, liver weight only increased from 0.9±0.1 g to 1.5±0.1 g, and hepatic triglyceride content increased from 43.7±26.5 mg/g to 104.9±62.1 mg/g after treated with T0901317. Though hepatic mRNA levels of FAS and SCD-1 enhanced, the hepatic mRNA level of FAS in NPC1L1-KO mice treated with T0901317 was still 63% lower than that in C57BL/6 mice T0901317 treatment. Conclusion The down-regulation of hepatic SREBP-1 and FAS induced by NPC1L1 elimination attenuates the LXR agonist-dependent hepatic steatosis in mice.