Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆, and thus, must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆, and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier-mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be: 1) linear with time for up to 10 min of incubation, and occurred with minimal metabolic alteration in the transported substrate, 2) temperature- and energy-dependent but Na⁺-independent in nature, 3) pH-dependent with higher uptake at acidic compared to alkaline buffer pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent K_m of 11.99 ± 1.41 µM and a V_max of 67.63 ± 3.87 pmol/mg protein/3 min, 5) inhibited by pyridoxine structural analogues (at buffer pH 5.5 but not 7.4), but not by unrelated compounds, 6) inhibited in a competitive manner by amiloride with an apparent K_i of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that while modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH-dependent and amiloride-sensitive, and appears to be under the regulation of an intracellular PKA-mediated pathway.