Endothelial-dependent coronary microvascular and vasomotor dysfunction (CMVD), which requires invasive coronary function test (ICFT) with intracoronary acetylcholine for diagnosis, is the predominant cause of angina in patients with no obstructive coronary artery disease (ANOCA). Oxygenation-sensitive cardiovascular magnetic resonance (OS-CMR) has been introduced as a non-invasive, needle-free diagnostic test for endothelium-dependent CMVD.This study aimed to compare OS-CMR with vasoactive breathing maneuvers with clinically performed, gold-standard ICFT in ANOCA patients with endothelial-dependent CMVD and healthy controls. We conducted a prospective, single-center study that enrolled 25 ANOCA patients diagnosed with endothelium-dependent CMVD by ICFT compared to 34 healthy participants. All participants underwent OS-CMR with vasoactive breathing maneuvers - inducing vasoconstriction then vasodilation. OS-CMR-derived biomarkers including the hyperventilation-induced myocardial oxygenation response (HV-MORE), breath-hold induced MORE (BH-MORE), and mean signal intensity (MI) were measured within the peri-systolic and late-diastolic phases of the cardiac cycle due to their physiologic relevance. Logistic regression with absolute shrinkage and selection operator (LASSO) penalization identified promising predictive biomarkers of CMVD in ANOCA patients.Compared to healthy participants, ANOCA-CMVD patients exhibited a significantly impaired oxygenation response to vasoactive breathing maneuvers. Late-diastolic HV-MOREMI-VAR and peri-systolic BH-MOREMI-AUC emerged as potential predictors of endothelial-dependent CMVD. The LASSO-derived multivariate model, adjusting for age, demonstrated apparent discrimination (AUC 0.92), sensitivity (88.9%), specificity (82.1%), positive predictive value (82.8%), negative predictive value (88.5%), and accuracy (85.5%) in identifying endothelial-dependent CMVD in ANOCA patients. In this small single-center cohort, we demonstrated the potential of physiologic markers derived from OS-CMR with vasoactive breathing maneuvers to differentiate ANOCA patients with ICFT confirmed endothelial-dependent CMVD from healthy controls. The clinical utility of this novel approach for non-invasively diagnosing CMVD needs further exploration with external validation in larger studies.