The vitamin folate is necessary for one-carbon metabolism in processes such as amino acid and nucleotide synthesis. As folate cannot be synthesized by mammals, it must be sourced from the diet and dietary supplements or generated by bacterial species present in the colon. There are a number of well-established adverse health outcomes associated with folate deficiency in humans, including congenital neural tube defects in newborns and colorectal cancer in adults. It has been proposed supra-physiological folate status can also be detrimental since it can lead to changes in immune function and the masking of vitamin B12 deficiency. It is generally believed that dietary sources of folate are primarily absorbed in the small intestine, however recent evidence from our team and others suggests that the colon may play a greater role in absorption of folate than previously understood. Specifically the amount of folate in the colon, much of it generated by the microbiome, can exceed dietary intakes of folate from natural sources. There are a number of gaps in knowledge regarding colonic folate absorption in humans. It is not known to what degree folate specific transporters such as proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) are present in the colon of healthy humans. To date, the majority of studies conducted identifying the presence of RFC and PCFT in colonocytes have been performed using cell lines or tissue harvested from organ donors. To our knowledge, no study has investigated the mRNA and protein concentrations of both RFC and PCFT present in human colonic tissue retrieved as biopsies. It also remains to be determined if folate status influences RFC and PCFT expression at the level of the colon. Secondly, it remains unclear to what degree the two intestinal folate hydrolases, glutamate carboxypeptidase II (GCPII) and γ-glutamyl hydrolase (GGH) are present in colonic mucosa. To gain a more thorough understanding of the fundamental processes involved in folate absorption in the colon of humans, the following three objectives are proposed. The primary objective is to evaluate the impact of 0 and 400 µg supplemental folic acid on total folate concentrations within the colonic mucosa by measuring folate levels. The secondary objectives include evaluating the impact of 0 and 400 µg supplemental folic acid on the regulation of PCFT and RFC in the ileum and colon by assessing the expression of mRNA transcripts and proteins, and determining the degree to which GCPII and GGH are present in the lumen of the ileum and colon by quantifying enzyme activity and expression of mRNA transcripts and proteins. To accomplish this, a 16-week longitudinal open-labelled randomized clinical trial will be conducted. Recruitment and screening will require three separate steps comprised of an initial invitation to participate, followed by a telephone screening session to determine eligibility and finally a baseline in-person study visit prior to enrollment into the intervention phase of the study. Once enrolled, participants will be asked to follow a diet low in synthetic folic acid for the duration of the study. At enrollment, subjects will be randomized to receive a multivitamin supplement and either 0 or 400 µg folic acid. Follow-up study visits will take place at week 8 and week 16 where anthropometric measurements, blood and colonic tissue biopsies (Week 16 only) from the terminal ileum, cecum, ascending colon, and descending colon will be collected for analysis. 24-hour dietary recalls will be collected throughout the study to observe folate and caloric intake. Plasma, RBC folate levels, and colonic mucosa folate content will be determined via microbial assay. GCPII and GGH enzyme activity will be determined according to the Krumdieck and Baugh method with modification. PCFT and RFC expression will be determined via semi-quantitative real-time PCR and protein samples will be extracted in a Western Blot Analysis using RIPA lysis and extraction buffer. The proposed study will further our understanding of the fundamental processes of folate metabolism and absorption in the colon. Results from this study may help to establish the optimal intakes of dietary folate, meeting the requirements of humans, without the potential health risks related to over-exposure to folate.