It is well-known that the pregnane X receptor (PXR/Nr1i2) is a critical xenobiotic-sensing nuclear receptor enriched in liver and intestine and is responsible for drug-drug interactions (DDI), due to their versatile ligand binding domain (LBD) and target genes involved in xenobiotic biotransformation. PXR can be modulated by various xenobiotics including pharmaceuticals, nutraceuticals, dietary factors, and environmental chemicals. Microbial metabolites such as certain secondary bile acids (BAs) and the tryptophan metabolite indole-3-propionic acid (IPA) are endogenous PXR activators. Gut microbiome is increasingly recognized as an important regulator for host xenobiotic biotransformation and intermediary metabolism. PXR regulates and is regulated by the gut-liver axis. This review summarizes recent research advancements leveraging pharmaco-and toxico-metagenomic approaches that have redefined the previous understanding of PXR. Key topics covered in this review include 1) genome-wide investigations on novel PXR-target genes, novel PXR-DNA interaction patterns, and novel PXR-targeted intestinal bacteria; 2) key PXRmodulating activators and suppressors of exogenous and endogenous sources; 3) novel bidirectional interactions between PXR and gut microbiome under physiological, pathophysiological, pharmacological, and toxicological conditions; and 4) modifying factors of PXR-signaling including species-and sex difference, and time (age, critical windows of exposure, and circadian rhythm). The review also discusses critical knowledge gaps and important future research topics centering around PXR.