Although studies have shown an inverse relationship between folate intake and colorectal cancer risk, the mechanisms by which folate deficiency may modulate colorectal cancer risk have not been established. An in vitro model of folate deficiency was used to determine the effects of folate deficiency on mRNA and/or protein expression of the genes involved in folate metabolism and one-carbon transfer reactions (GCP-II, FR, RFC, FPGS, GGH, MTR, MTRR, MTHFR, DHFR, TS, cSHMT, DNMT-1, -3a, -3b and DeMTase). The results showed complex and, in some cases, cell-specific effects of folate deficiency on mRNA and/or protein expression of these genes. This study provides a frame work for future studies investigating the modulatory effects of folate deficiency on the proteins and enzymes involved in folate metabolism and one-carbon transfer reactions.