The mechanism of uptake of dietary niacin (nicotinic acid) by intestinal epithelial cells is not well understood, and nothing is known about regulation of the uptake process. In this investigation, we used the human-derived intestinal epithelial Caco-2 cells and purified intestinal brush border membrane vesicles (BBMV) isolated from human organ donors to assess niacin uptake. Our findings showed niacin uptake by Caco-2 cells to be: 1) temperature- and energy-dependent; 2) Na⁺ independent but highly dependent on extracellular acidic pH; 3) saturable as a function of concentration with an apparent K_m of 0.53 ± 0.08 µM; 4) severely inhibited by the membrane impermeable sulfhydryl group reagents; and 5) highly specific for niacin but not affected by monocarboxylic acids. A marked trans-stimulation in [³H]-niacin efflux from preloaded Caco-2 cells by unlabeled niacin in the incubation buffer was also observed. These findings suggest the involvement of a specialized, pH dependent, carrier-mediated mechanism for human intestinal niacin uptake. This suggestion was confirmed in studies with native human intestinal BBMV. We also examined possible regulation of niacin uptake by Caco-2 cells by specific intracellular regulatory pathways. The results showed that while the PKA-, PKC- and Ca/calmodulin- mediated regulatory pathways play no role in regulating niacin uptake, a role for protein tyrosine kinase (PTK)-mediated pathway was apparent. Results of these studies show for the first time the existence of a specialized, acidic pH-dependent, carrier-mediated system for niacin uptake by human intestinal epithelial cells that operates at the micromolar (physiological) range of niacin. The results also suggest possible involvement of a PTK-mediated pathway in the regulation of niacin uptake.