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Objectives: We investigated the relationship between low wall shear stress (WSS) and severe endothelial dysfunction (EDFx). Background: Local hemodynamic forces, such as WSS play an important role in atherogenesis through their effect on endothelial cells. We hypothesized that low WSS independently predicts severe EDFx in patients with coronary artery disease (CAD). Methods: Forty-four patients with CAD underwent coronary angiography, fractional flow reserve (FFR) and endothelial function testing. Segments with >10% vasoconstriction after acetylcholine (Ach) infusion were defined as having severe EDFx. WSS, calculated using 3D angiography, velocity measurements and computational fluid dynamics, was defined as low (< 1 Pa), intermediate (1–2.5 Pa) or high (> 2.5 Pa). Results: Median age was 52 years, 73% were females. Mean FFR was 0.94 ± 0.06. In 4,510 coronary segments, median WSS was 3.67 Pa. 24% had severe EDFx. A higher proportion of segments with low WSS had severe EDFx (71%) compared to intermediate WSS (22%) or high WSS (23%) (p < 0.001). Segments with low WSS demonstrated greater vasoconstriction in response to ACh than intermediate or high WSS segments (−10.7% vs. −2.5% vs. +1.3%, respectively, p < 0.001). In a multivariable logistic regression analysis, female sex (OR: 2.44, p = 0.04), diabetes (OR: 5.01, p = 0.007) and low WSS (OR: 9.14, p < 0.001) were independent predictors of severe EDFx. Conclusion: In patients with non-obstructive CAD, segments with low WSS demonstrated more vasoconstriction in response to ACh than intermediate or high WSS segments. Low WSS was independently associated with severe endothelial dysfunction.