In humans and rodents, phenobarbital (PB) induces hepatic and extra-hepatic drug metabolizing enzymes (DMEs) through the activation of specific nuclear receptors (NRs). In contrast, few data about PB transcriptional effects in veterinary species are available. The constitutive expression and modulation of PB-responsive NR and DME genes, following an oral PB challenge, were investigated in cattle liver and extra-hepatic tissues (duodenum, kidney, lung, testis, adrenal and muscle). Likewise to humans and rodents, target genes were expressed to a lower extent compared to the liver with few exceptions. Phenobarbital significantly affected hepatic CYP2B22, 2C31, 2C87, 3A and UDP-glucuronosyltransferase 1A1-like, glutathione S-transferase A1-like and sulfotransferase 1A1-like (SULT1A1-like) mRNAs and apoprotein amounts; in extra-hepatic tissues, only duodenum showed a significant down-regulation of SULT1A1-like gene and apoprotein. Nuclear receptor mRNAs were never affected by PB. Presented data are the first evidence about the constitutive expression of foremost DME and NR genes in cattle extra-hepatic tissues, and the data obtained following a PB challenge are suggestive of species-differences in drug metabolism; altogether, these information are of value for the extrapolation of pharmacotoxicological data among species, the characterization of drug-drug interactions as well as the animal and consumer's risk caused by harmful residues formation.