The vitamin D₃-25-hydroxylase CYP27A is located predominantly in liver, but its expression is also detected in extrahepatic tissues. Our aim was to evaluate the regulation of CYP27A by vitamin D₃ (D₃) or its metabolites in rat duodena. Vitamin D-depleted rats were repleted with D₃, 25-hydroxyvitamin D (25OHD), or 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] or acutely injected 1,25(OH)₂D₃ to investigate the mechanisms of action of the hormone. All D₃ compounds led to a progressive decrease in CYP27A mRNA, with levels after D₃ representing 20% of that observed in D depletion. 25OHD decreased CYP27A mRNA by 55%, whereas 1,25(OH)₂D₃ led to a 40% decrease, which was accompanied by a 31% decrease in CYP27A protein levels and an 89% decrease in enzyme activity. Peak circulating 1,25(OH)₂D₃ concentrations were, however, the highest in D₃-repleted, followed by 25OHD- and 1,25(OH)₂D₃-repleted animals. 1,25(OH)₂D₃ resulted in a decrease in both CYP27A mRNA half-life and transcription rate. Our data illustrate that the intestine expresses the D₃-25-hydroxylase and that the gene is highly regulated in vivo through a direct action of 1,25(OH)₂D₃ or through the local production of D₃ metabolites.