Rilpivirine and etravirine are second generation non-nucleoside reverse transcriptase inhibitors approved recently by the United States Food and Drug Administration for the treatment of human immunodeficiency virus-1 infection. Pregnane X receptor (PXR) is a member of the superfamily of nuclear receptors that regulate the expression of various genes controlling diverse biological functions. The present study investigated the effects of rilpivirine and etravirine on the activity of human PXR (hPXR), including the mode of activation, and compared them to those of efavirenz, nevirapine, and delavirdine, which are first generation non-nucleoside reverse transcriptase inhibitors. In transiently transfected HepG2 cells, rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, activated human, mouse, and rat PXR. Results from mechanistic studies indicated that rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, bound to the ligand-binding domain of hPXR, as assessed by a transactivation assay and by a competitive ligand-binding assay using time-resolved fluorescence resonance energy transfer; triggered nuclear translocation of a green fluorescence protein-tagged hPXR, as visualized by confocal imaging; and recruited steroid receptor coactivator-1 (SRC-1), SRC-2, and SRC-3 to hPXR, as demonstrated by mammalian two-hybrid assays. Rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, increased hPXR target gene (CYP3A4) expression in primary cultures of human hepatocytes. In summary, select non-nucleoside reverse transcriptase inhibitors activated human and rodent PXR. Rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, were identified as agonists of hPXR, as assessed in mechanistic experiments, and inducers of CYP3A4, as determined in primary cultures of human hepatocytes.