The human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and II drug-metabolizing enzymes as well as that of drug transporters. In addition, this receptor plays a critical role in cholesterol homeostasis and in protecting tissues from potentially toxic endobiotics. hPXR is activated by a broad spectrum of low-affinity compounds including xenobiotics and endobiotics such as bile acids and their precursors. Crystallographic studies revealed a ligand binding domain (LBD) with a large and conformable binding pocket that is likely to contribute to the ability of hPXR to respond to compounds of varying size and shape. Here, we describe an in silico method that allowed the identification of nine novel hPXR agonists. We further characterize the compound 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide (C2BA-4), a methanesulfonamide that activates PXR specifically and more potently than does the reference compound 4-[2,2-bis(diethoxyphosphoryl)ethenyl]-2,6-ditert-butyl-phenol (SR12813) in our stable cell line expressing a Gal4-PXR and a GAL4 driven luciferase reporter gene. Furthermore treatment of primary human hepatocytes with C2BA-4 results in a marked induction of the mRNA expression of hPXR target genes, such as cytochromes P450 3A4 and 2B6. Finally, C2BA-4 is also able to induce hPXR-mediated in vivo luciferase expression in HGPXR stable bioluminescent cells implanted in mice. The study suggests new directions for the rational design of selective hPXR agonists and antagonists.