<b>Background:</b> Microvascular angina is associated with dysregulation of the endothelin system and impairments in myocardial blood flow, exercise capacity, and health-related quality of life. The G allele of the noncoding single nucleotide polymorphism <i>RS9349379</i> enhances expression of the endothelin-1 gene (<i>EDN1</i>) in human vascular cells, potentially increasing circulating concentrations of Endothelin-1 (ET-1). Whether zibotentan, an oral <i>ET-A</i> receptor selective antagonist, is efficacious and safe for the treatment of microvascular angina is unknown. <b>Methods:</b> Patients with microvascular angina were enrolled in this double-blind, placebo-controlled, sequential crossover trial of zibotentan (10 mg daily for 12 weeks). The trial population was enriched to ensure a G allele frequency of 50% for the <i>RS9349379</i> single nucleotide polymorphism. Participants and investigators were blinded to genotype. The primary outcome was treadmill exercise duration (seconds) using the Bruce protocol. The primary analysis estimated the mean within-participant difference in exercise duration after treatment with zibotentan versus placebo. <b>Results:</b> A total of 118 participants (mean ±SD; years of age 63.5 [9.2 ]; 71 [60.2% ] females; 25 [21.2% ] with diabetes) were randomized. Among 103 participants with complete data, the mean exercise duration with zibotentan treatment compared with placebo was not different (between-treatment difference, -4.26 seconds [95 ] CI, -19.60 to 11.06] <i>P</i>=0.5871). Secondary outcomes showed no improvement with zibotentan. Zibotentan reduced blood pressure and increased plasma concentrations of ET-1. Adverse events were more common with zibotentan (60.2%) compared with placebo (14.4%; <i>P</i><0.001). <b>Conclusions:</b> Among patients with microvascular angina, short-term treatment with a relatively high dose (10 mg daily) of zibotentan was not beneficial. Target-related adverse effects were common.