5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme in the folate metabolic pathway that contributes to DNA, protein and small molecule methylation. Previous studies have linked common genetic polymorphisms in the human MTHFR gene to hyperhomocysteinemia, a risk factor for cardiovascular disease. However, the full range of common genetic polymorphisms and haplotypes in the human MTHFR gene remains unclear. Furthermore, the underlying cellular mechanisms by which common nonsynonymous coding single nucleotide polymorphisms (cSNPs) might alter the function of this enzyme are not defined. The present study focused on the systematic identification and investigation of common polymorphisms in the MTHFR gene using a genotype-to-phenotype approach followed by studies of their molecular mechanisms for these functional effects. Specifically, we began by resequencing exons, splice junctions and portions of the 5′-flanking region of the human MTHFR gene using DNA samples from 60 African-American, 60 Caucasian-American, 60 Han-Chinese American, and 60 Mexican-American subjects obtained from the Coriell Cell Repository. A total of 66 polymorphisms, 63 SNPs and 3 insertion/deletion events, were observed. Ten nonsynonymous cSNPs were identified, including the known C677T (A222V) and A1298C (E429A) variants. We then performed functional genomic studies with all of the nonsynonymous cSNPs observed. Specifically, the wild-type (WT) and 10 variant allozymes were transiently expressed in COS-1 cells. After correction for transfection efficiency, activity for the variant allozymes ranged from 6.5% to 145% of WT activity. Levels of immunoreactive protein for the allozymes ranged from 15% to 118% of WT and were significantly correlated with levels of enzyme activity (Rp = .93, p = .0001), suggesting that a major mechanism by which nonsynoymous cSNPs influence the function of this gene is by alteration in the quantity of protein. These observations represent steps toward an understanding of molecular genetic mechanisms responsible for variation in MTHFR levels and/or properties that may contribute to the pathophysiology of human disease.