The pancreas contains the second highest level of folate (after the liver) among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanism(s) involved in folate uptake by cells of this important exocrine organ, and its regulation. We have begun to address these issues and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results showed folic acid uptake to be: 1) temperature- and energy-dependent, 2) pH-dependent with a markedly higher uptake at acidic pH compared to neutral or alkaline pHs, 3) Na⁺-independent, 4) saturable as a function of substrate concentration (apparent K_m= 0.762 ± 0.10 µM), 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives, 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. Functional contribution of the hRFC in carrier-mediated folate uptake was confirmed by mean of gene silencing using gene-specific siRNA. Evidence was also obtained to suggest that the folate uptake process by MIA PaCa-2 cells is under the regulation of cAMP- and protein tyrosine kinase (PTK)- mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent carrier-mediated mechanism for folate uptake by the human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process, and suggest possible involvement of an intracellular cAMP and PTK-mediated pathways in the regulation of folate uptake.