Objective: To examine the efficacy and effect modifiers of folic acid supplementation in the prevention of stroke in regions without folic acid fortification based on relevant, up-to-date published randomized trials. Methods: Relative risk (RR) was used to measure the effect of folic acid supplementation on risk of stroke using a fixed effects model. Findings: Overall, folic acid supplementation significantly reduced the stroke risk by 11%(22 trials, n = 82,723; RR 0.89, 95% confidence interval [CI] 0.84-0.96). The effect was greater in low folate regions (2 trials, n = 24,020; Asia, 0.78, 0.67-0.90) compared to high folate regions (7 trials, n = 14,655; America, 1.05, 0.90-1.23), and among patients without folic acid fortification (11 trials, n = 49,957; 0.85; 0.77-0.94) compared with those with folic acid fortification (7 trials, n = 14,655; 1.05, 0.90-1.23). In further stratified analyses among trials without folic acid fortification, a larger beneficial effect was found in those trials that used a low dosage of folic acid (<= 0.8 mg: 0.78, 0.69-0.88) or low baseline vitamin B-12 levels (<384 pg/mL: 0.78, 0.68-0.89). In the corresponding comparison groups, the effect sizes were attenuated and insignificant (p for interaction <0.05 for both). Although the interaction tests were not significant, there might be a higher benefit in trials with a low dosage of vitamin B-12, a low prevalence of statin use, but a high prevalence of hypertension. Conclusions: Folic acid supplementation could reduce the stroke risk in regions without folic acid fortification, particularly in trials using a relatively low dosage of folic acid and with low vitamin B12 levels.