Pregnane X receptor (PXR) is known as a xenosensor, playing a key role in response to xenochemical stimuli. Activation of PXR enhanced expression of various drug-metabolizing enzymes and transporters such as cytochrome P450 3A4 (CYP3A4). During a screening of a natural compounds library for novel ligands of human xenosensing receptors by the mammalian one-hybrid assay, two cyclohexene-type amide alkaloids were isolated, with nigramide C (NigC) showing the most potent activation of human PXR (hPXR). NigC-mediated hPXR activation was enhanced by overexpression of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor γ, coactivator 1α, and protein arginine methyltransferase 1. A direct interaction between the ligand-binding domain of hPXR and the receptor interaction domain of SRC1 was observed. NigC induced the expression of endogenous CYP3A4 mRNA and protein in both cultured hepatoma cells and primary hepatocytes. However, in primary hepatocytes, the relative agonist activity of NigC was not as potent as that of rifampicin, probably because of lower metabolic stability of NigC in these cells. In conclusion, NigC was found to be an effective agonist of hPXR. NigC is a useful tool for investigation of hPXR function.