Background. Standard of care for chronic spontaneous urticaria (CSU) includes second-generation H1-antihistamines (SGAH) but is often ineffective even with four-times the FDA-recommended dose. Urticarial lesions are commonly characterized by increased lymphocytes with perivascular eosinophilic infiltrates implying a role for the interleukin-5 (IL5). Objective. This study investigated the effects of benralizumab, an anti-IL5-receptor-alpha monoclonal antibody, in human subjects with SGAH-unresponsive CSU that completed all study visits. Methods. A repeated-measures, 24-week study was conducted at an urticaria specialist clinic where SGAH-unresponsive CSU patients (3 males and 9 females; age range, 32-65 years) having a median weekly Urticaria Activity Score (UAS7) of 4 and pruritus severity ≥2 were enrolled. After a baseline run-in period, subjects were treated with a subcutaneous placebo dose followed by benralizumab 30mg subcutaneously every month (×3 doses) followed by two off-medication monthly-visits. The primary and exploratory endpoints were the change from baseline in UAS7 and Chronic Urticaria Quality-of-Life Score (CU-QoLTS) respectively. Secondary endpoints included peripheral blood eosinophils (eos%) and basophils, skin eosinophilia, and differentially expressed genes (DEGs) in blood before- and after benralizumab. Results. UAS7 and CU-QoLTS significantly improved post-benralizumab compared to baseline scores in 7 of 9 subjects completing the study. Clinical improvements correlated with reduction in eos% and inflammatory cell infiltrates in skin lesions. Biologic pathways, regulated by DEGs, involved IL-5R activity, tryptophan metabolism and Siglec-8 expression. Conclusion. Benralizumab was clinically efficacious in the treatment of subjects with SGAH-refractory CSU which correlated with several DEGs in blood. This study supports the use of benralizumab for CSU treatment.