Ileal Apical Sodium-Dependent Bile Acid Transporter (ASBT) is responsible for reabsorbing the majority of bile acids from the intestinal lumen. Rapid adaptation of ASBT function in response to physiological and pathophysiological stimuli is essential for the maintenance of bile acid homeostasis. However, not much is known about molecular mechanisms responsible for acute post-transcriptional regulation of ileal ASBT. Protein kinase C (PKC)-dependent pathway represents a major cell signaling mechanism influencing intestinal epithelial functions. The present studies were, therefore, undertaken to investigate ASBT regulation in intestinal Caco2 monolayers by the well-known PKC activator phorbol ester PMA. Our results showed that sodium-dependent ³H-TC uptake in Caco2 cells was significantly inhibited in response to 2 h incubation with 100 nM PMA compared to incubation with 4PMA (inactive form). The inhibitory effect of PMA was blocked in the presence of 5 µM Bisindolylmaleimide I (protein kinase C inhibitor) but not BAPTA-AM (Ca⁺⁺ chelator) or LY294002 (PI3K inhibitor). PMA inhibition of ASBT function was also abrogated in the presence of myristoylated PKC pseudosubstarte peptide indicating involvement of atypical PKC. The inhibition by PMA was associated with a significant decrease in the V_max of the transporter and a reduction in ASBT plasma membrane content suggesting a modulation by vesicular recycling. Our novel findings demonstrate a post-transcriptional modulation of ileal ASBT function and membrane expression by phorbol ester via PKC-dependent pathway.