Genes functioning in folate-mediated 1-carbon metabolism are hypothesized to play a role in cardiovascular disease (CVD) risk beyond the current narrow focus on the MTHFR 677 C→T (rs1801133) polymorphism. Using a cohort study design, we investigated whether sequence variants in the network of folate-related genes, particularly in genes encoding proteins related to SHMT1, predict CVD risk in 1131 men from the Normative Aging Study. A total of 330 single nucleotide polymorphisms (SNPs) in 52 genes, selected for function and gene coverage, were assayed on the Illumina GoldenGate platform. Age- and smoking-adjusted genotype-phenotype associations were estimated in regression models. Using a nominal P ≤ 5.00 × 10(-3) significance threshold, 8 SNPs were associated with CVD risk in single locus analyses. Using a false discovery rate (FDR) threshold (P-adjusted ≤1.00 × 10(-1)), a SNP in the GGH gene remained associated with reduced CVD risk, with a stronger association in early onset CVD cases (<55 y). A gene × folate interaction (MAT2B) and 2 gene × vitamin B-12 interactions (BHMT, SLC25A32) reached the FDR P-adjusted ≤2.00 × 10(-1) threshold. Three biological hypotheses related to SHMT1 were explored and significant gene × gene interactions were identified for TYMS by UBE2N, FTH1 by CELF1, and TYMS by MTHFR. Variations in genes other than MTHFR and those directly involved in homocysteine metabolism are associated with CVD risk in non-Hispanic white males. This work supports a role for SHMT1-related genes and nuclear folate metabolism, including the thymidylate biosynthesis pathway, in mediating CVD risk.