Introduction: Diamine oxidase (DAO) catabolizes and inactivates histamine, a key player in a wide range of invalidating conditions, such as migraine and chronic spontaneous urticaria (CSU). The highest expression of DAO occurs in the gastrointestinal tract, possibly to control the burden of histamine intake from food. Methods: Here, we tested the hypothesis that a 30-day oral supplementation with DAO (1 capsule b.i.d., 15 min before a meal) could reduce the severity of CSU as estimated by the 7-Day Urticaria Activity Score (UAS-7). The study was designed as a double-blind, placebo-controlled, crossover investigation of 22 patients with CSU incompletely controlled by first-line antihistamine therapy. Results: Twenty patients completed the study. Supplemental therapy with DAO caused a 3.8 ± 1.2 point mean ± SEM UAS-7 score reduction in patients with low serum DAO levels at time 0 (p = 0.041 compared to placebo). The degree of UAS-7 improvement was inversely correlated with the levels of basal DAO (p = 0.019). Patients receiving DAO supplementation were able to slightly reduce their daily antihistamine dose (p = 0.049). Conclusion: These data suggest that DAO may be involved in the pathogenic cascade of CSU and that DAO supplementation could be effective for symptom relief in patients with low DAO levels in serum.