Flow-mediated dilation (FMD) is a noninvasive index of endothelial function and vascular health in humans. Studies examining the role of nitric oxide (NO) are not conclusive. In this article, we quantified the contribution of NO in FMD of conduit arteries and explored the effect of the protocol (ie, distal cuff, ≈5-minute ischemia) and method of analysis (ie, automated and continuous edge detection) on the NO dependency of this test. A systematic review and 3-stage meta-analysis of published crossover studies that measured FMD under local infusion of saline or the NO synthase blocker N^Gmonomethyl-L-arginine (L-NMMA) was undertaken. Twenty studies met the inclusion criteria for stage 1 (374 individual comparisons). The meta-analyzed outcome was the difference in FMD between infusion of saline (ie, FMD_saline) and NO synthase blocker (ie, FMD_L-NMMA). Overall, FMD_saline was 8.2% (95% confidence interval [CI], 6.8%–9.6%) compared with FMD_L-NMMA of 3.7% (95% CI, 3.1%–4.3%; P<0.001). Stage 2 analysis focused on studies that used the most commonly adopted approach in healthy volunteers (ie, distal cuff placement, ≈5-minute occlusion), which similarly revealed a significant NO contribution to FMD (FMD_saline, 6.5% [95% CI, 5.7%–7.3%]; FMD_L-NMMA, 0.9% [95% CI, 0.5%–1.3%]; P<0.001). Stage 3 meta-analyzed the studies that adopted the commonly adopted approach and automated, continuous method of analysis, which also revealed a significant contribution of NO to the FMD (FMD_saline, 6.9% [95% CI, 6.0%–7.8%]; FMD_L-NMMA, 2.4% [95% CI, 1.1%–3.7%]; P<0.001). This comprehensive analysis demonstrates that FMD of conduit arteries in humans is, at least in part, mediated by NO.