Folic acid is a vitamin essential for thymidylate and purine synthesis. The human proton-coupled folate transporter (hPCFT) has recently been identified as a pH-dependent folic acid transporter and mutations in this transporter have been linked to hereditary folic acid malabsorption. In this study we assessed hPCFT mediated transport activity in vitro, intersubject variability of intestinal expression in relation to blood folates and the relationship of proton-pump inhibitor (PPI) therapy on hPCFT expression in vivo. We created an MDCKII cell line stably expressing hPCFT to evaluate its drug substrates and inhibitors. Intestinal pinch biopsies (duodenum, ileum, colon) were collected from patients undergoing routine endoscopy procedures and expressed levels of hPCFT were determined by RT-PCR. When assessed using MDCKII-hPCFT cells, folic acid and methotrexate were found to be high affinity hPCFT substrates. Sulfasalazine and pyrimethamine were noted to inhibit hPCFT activity with Ki values of 42.3 and 161.7 µmol/L, respectively. hPCFT was localized to the brush border membrane of enterocytes with highest expression in the duodenum and reduced levels in the ileum and colon. When we assessed hPCFT expression in a subset of patients who were receiving PPI therapy, a near 50% reduction in duodenal hPCFT mRNA expression was noted. These results suggest that hPCFT transporter activity can be modulated by many drugs in clinical use, and expression of this transporter in the GI tract is higher in the duodenum than more distal sites (duodenum>ileum>colon). Importantly we note that PPI drug use appears to be associated with reduced hPCFT expression in vivo.