Pregnane X receptor (PXR) mainly regulates xenobiotic metabolism and detoxification. Additionally, it exerts pleiotropic effects on liver physiology, which in large parts depend on transrepression of other liver-enriched transcription factors. Based on the hypothesis that lower expression levels of PXR may reduce the extent of this inhibition, an exploratory genome-wide transcriptomic profiling was performed using HepG2 cell clones with different expression levels of PXR. This screen and confirmatory real-time RT-PCR identified sphingomyelin phosphodiesterase acid-like (SMPDL) 3A, a novel nucleotide phosphodiesterase and phosphoramidase, as being up-regulated by PXR-deficiency. Transient siRNA-mediated knock-down of PXR in HepG2 cells and primary human hepatocytes similarly induced mRNA up-regulation, which translated into increased intracellular and secreted extracellular protein levels. Interestingly, ligand-dependent PXR activation also induced SMPDL3A in HepG2 cells and primary human hepatocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of PXR to the previously identified liver X receptor (LXR)-binding DR4 motif as well as to an adjacent ER8 motif in intron 1 of SMPDL3A. Constitutive binding of the unliganded receptor to the intron 1 chromatin indicated ligand-independent repression of SMPDL3A by PXR. Transient transfection and reporter gene analysis confirmed the specific role of these motifs in PXR- and LXR-dependent activation of the SMPDL3A intronic enhancer. PXR inhibited LXR mainly by competition for binding sites. In conclusion, this study describes that a decrease in PXR expression levels and ligand-dependent activation of PXR and LXR increase hepatic SMPDL3A levels, which possibly connects these receptors to hepatic purinergic signaling and phospholipid metabolism and may result in drug-drug interactions with phosphoramidate pro-drugs.