ObjectiveThe aim of this study was to investigate the relationship between 25-hydroxyvitamin D3 (25[OH]D3) and ischemic stroke and its potential modifying factors in rural Chinese adults. MethodsThis nested case-control study was drawn from the H-type Hypertension and Stroke Prevention and Control Project, a community-based, prospective, observational study. Plasma 25(OH)D3 was measured by liquid chromatography with tandem quadrupole mass spectrometry. All stroke records came from the Chinese Center for Disease Control and Prevention. Multiple logistic regression models were used to evaluate the association between 25(OH)D3 and risk of ischemic stroke. ResultsWe included 1079 participants with ischemic stroke and 1079 matched controls. Due to a non-linear relationship, the analyses were stratified by 25(OH)D3. For those with 25(OH)D3 < 20 ng/mL, there was a 15% reduction in the risk of ischemic stroke for each SD increment in 25(OH)D3 (odds ratio, 0.85; 95% confidence interval, 0.73–0.99). Compared with the lowest-tertile group, the risk of ischemic stroke decreased by 39% (odds ratio, 0.61; 95% confidence interval, 0.41–0.89) in the highest-tertile group. Furthermore, two effect modifiers were identified: diabetes and homocysteine level. Although participants with 25(OH)D3 ≥ 20 ng/mL had the lowest risk of ischemic stroke overall, there was no dose-response association within that range. ConclusionsAn inverse dose-response association between 25(OH)D3 and incident risk of ischemic stroke in rural Chinese adults was only observed in those with 25(OH)D3 < 20 ng/mL, along with two effect modifiers. Higher levels of 25(OH)D3 did not confer additional benefit.