Folate, a water-soluble B vitamin and cofactor in one-carbon transfer, is an important nutritional factor that may modulate the development of colorectal cancer (CRC). Epidemiologic and clinical studies indicate that dietary folate intake and blood folate levels are inversely associated with CRC risk. Collectively, these studies suggest an ∼ 40% reduction in the risk of CRC in individuals with the highest dietary folate intake compared with those with the lowest intake. Animal studies using chemical and genetically predisposed rodent models have provided considerable support for a causal relationship between folate depletion and colorectal carcinogenesis as well as a dose-dependent protective effect of folate supplementation. However, animal studies also have shown that the dose and timing of folate intervention are critical in providing safe and effective chemoprevention; exceptionally high supplemental folate levels and folate intervention after microscopic neoplastic foci are established in the colorectal mucosa promote, rather than suppress, colorectal carcinogenesis. These animal studies, in conjunction with clinical observations, suggest that folate possesses dual modulatory effects on carcinogenesis depending on the timing and dose of folate intervention. Folate deficiency has an inhibitory effect, whereas folate supplementation has a promoting effect on the progression of established neoplasms. In contrast, folate deficiency in normal epithelial tissues appears to predispose them to neoplastic transformation, and modest levels of folate supplementation suppress the development of tumors in normal tissues. Notwithstanding the limitations associated with animal models, these studies suggest that the optimal timing and dose of folate intervention must be established for safe and effective chemoprevention in humans. Folate is an important factor in DNA synthesis, stability, and integrity, the repair aberrations of which have been implicated in colorectal carcinogenesis. Folate may also modulate DNA methylation, which is an important epigenetic determinant in gene expression (an inverse relationship), in the maintenance of DNA integrity and stability, in chromosomal modifications, and in the development of mutations. A mechanistic understanding of how folate status modulates colorectal carcinogenesis further strengthens the case for a causal relationship and provides insight into a possible chemopreventive role of folate. Environ. Mol. Mutagen. 44:10–25, 2004. © 2004 Wiley-Liss, Inc.