Hepatic steatosis is a major metabolic concern associated with activation of the pregnane X receptor (PXR), a nuclear receptor known to promote lipogenesis and lipid accumulation in the liver. While the lipogenic effects of PXR agonists are well documented, there is no comprehensive data on the metabolic consequences of PXR inhibition. Here, we evaluated the impact of MI-891, a novel human PXR antagonist and inverse agonist, on hepatic lipid metabolism in high-fat diet (HFD)-fed PXR-CAR-CYP3A4/3A7-humanized female mice. Lipidomic analysis revealed a significant reduction in hepatic triglyceride levels and a shift in their saturation profile after MI-891 treatment. In parallel, RT-qPCR analysis showed a down-regulation of lipogenic genes ( Scd1 , Thrsp , Acaca ) and lipid-droplet-associated genes ( Plin2 , Hsd17b13 ), suggesting suppressed fatty acid and triglyceride synthesis and diminished lipid droplet storage. The primary PXR target gene CYP3A4 was downregulated at both the mRNA and protein levels, confirming PXR inhibition in vivo . Our findings demonstrate that MI-891 treatment effectively inhibits PXR activity and leads to significant alterations in the hepatic lipidome and lipid metabolism under HFD conditions. This study represents the first report of liver lipidomics in response to a PXR inhibitor, highlighting PXR inhibition as a potential therapeutic approach for hepatic steatosis.