Folate cofactors play crucial roles in hundreds of reactions in cells including DNA and protein synthesis. The human proton-coupled folate transporter (PCFT) is the only means of absorption of dietary folates in humans. PCFT expression and function is associated with many disorders including hereditary folate malabsorption, neural tube defects, Down syndrome, cancer, heart diseases, Alzheimer's and Parkinson's disease. Upregulation of PCFT expression in tumor cells is of significant consideration for development of PCFT-targeted chemotherapeutic agents. However, not much is known about the structure and function of PCFT, which contributes to the low clinical success rate of folate-based agents. To address this gap in the knowledge we performed extensive Cys-mutagenesis studies. We analyzed 40 residues towards the extracellular face of PCFT, 35 positions towards the cytoplasmic face of PCFT and 28 positions along the proposed folate-binding pocket of PCFT. Based on the accessibility studies of the extracellular face of PCFT we determined loop-helix boundaries of this face and identified the glycerol-3-phosphate transporter (PDB#1PW4) and tripeptide-proton symporter (PDB#4APS) as the best templates for modeling PCFT. Based on the accessibility studies of the cytoplasmic face of PCFT, we identified loop-helix boundaries of this face. Here, we also show that our accessibility studies support the hypothesis that PCFT is present predominantly in an inward-open conformation in the absence of substrate (pH 7.5 and no folic acid). We also show that the folate-binding pocket of PCFT is formed by residues present in PCFT transmembrane helices I, IV, V, X and XI. Our results are of high significance in understanding the details of folate-homeostasis mechanisms and in design of PCFT-targeted therapeutic and diagnostic agents.