Abstract Objective We evaluated the interaction of serum folate and vitamin B12 (B12) with methylenetetrahydrofolate reductase (MTHFR) C677T genotypes, on the risk of first ischemic stroke, and on the efficacy of folic acid (FA) treatment in prevention of first ischemic stroke. Methods A total of 20,702 hypertensive adults were randomized to a double-blind treatment of daily enalapril 10mg and FA 0.8mg or enalapril 10mg alone. Participants were followed-up every three months. Results Median values of folate and B12 concentrations at baseline were 8.1 ng/mL and 280.2 pmol/L, respectively. Over a median of 4.5 years, among those not receiving FA, participants with baseline serum B12 and/or serum folate above the median had a significantly lower risk of first ischemic stroke (HR, 0.74; 95%CI: 0.57-0.96), especially in those with MTHFR 677 CC genotype (wild type) (HR, 0.49; 95%CI: 0.31-0.78). FA treatment significantly reduced the risk of first ischemic stroke in participants with both folate and B12 below the median (2.3% in enalapril-folic acid group vs. 3.6% in enalapril-only group; HR, 0.62; 95%CI: 0.46-0.86), particularly in MTHFR 677 CC carriers (1.6% vs. 4.9%; HR, 0.24; 95%CI: 0.11-0.55). However, carriers of TT responded better with both folate and B12 levels above the median (HR, 0.28; 95%CI: 0.10-0.75). Conclusions The risk of first ischemic stroke was significantly higher in hypertensive patients with low levels of both folate and B12. Effect of FA treatment was greatest in patients with low folate and B12 with the CC genotype, and with high folate and B12 with the TT genotype.