Meta-analyses have suggested an effect of MTHFR C677T genotype (rs1801133), a proxy for blood total homocysteine, on cardiovascular disease (CVD) in populations with low population dietary folate. The aim was to examine the association and effect modification by serum folate and vitamin B12 levels between MTHFR and CVD-related outcomes in a general population with no mandatory folic acid fortification policy.The study population included 13,748 adults retrieved from pooling of four population-based studies conducted in Denmark. MTHFR genotype, serum folate (measured in approximately 9,356 individuals), and serum vitamin B12 (9,215 individuals), hypertension, and dyslipidemia were measured at baseline, and participants were followed for a mean of 10.5-11.7 years in central registries for diagnoses of stroke (623 incidents), ischaemic heart disease (IHD) (835 incidents), and all-cause mortality (1,272 incidents).The MTHFR genotype (TT vs. CC/CT) was not associated with hypertension [OR (95% CI) 1.09 (0.95-1.25)], dyslipidemia [OR (95% CI) 0.97 (0.84-1.11)], stroke [HR (95% CI) 0.92 (0.69-1.23)], and all-cause mortality [HR (95% CI) 0.94 (0.77-1.14)], either overall, or in participants with low serum folate or B12 status (P values for interactions 0.15-0.94). Individuals with the MTHFR TT genotype had a higher risk of IHD (HR (95% CI) 1.38 (1.11-1.71)), but this association was not modified by folate status (P value for interaction 0.45).Our results do not support a causal relationship between homocysteine and CVD. However, we cannot exclude a direct causal effect of MTHFR C677T genotype on IHD.