Summary Background High‐mobility group box 1 protein (HMGB1) belonging to endogenous danger signals prolongs eosinophil survival and acts as a chemoattractant. Objective The authors evaluated the role of HMGB1 in the pathogenesis of asthma characterized by eosinophilic airway inflammation. Methods Firstly, HMGB1 expressions in induced sputum obtained from human asthmatics were determined. This was followed by an evaluation of the role of HMGB1 in a murine model of asthma using anti‐HMGB1 antibodies. Then the effect of HMGB1 on the receptor of advanced glycation end products (RAGE) expressions on CD11b‐CD11c+ cells isolated from a murine model of asthma were measured to elucidate the mechanisms involved. Results Sputum HMGB1 expressions were markedly higher in asthmatics than in normal controls, and were positively correlated with sputum eosinophilia and sputum TNF‐α, IL‐5 and IL‐13 expressions. In a murine model of asthma, HMGB1 expressions in lung tissue and HMGB1 levels in bronchoalveolar lavage fluid were significantly elevated and eosinophilic airway inflammation, non‐specific airway hyperresponsiveness, and pathological changes were attenuated by blocking HMGB1 activity. Furthermore, we found that enhanced RAGE expressions on CD11b‐CD11c+ also significantly decreased when HMGB1 activity was blocked. Conclusion and Clinical Relevance Our findings suggest that HMGB1 plays a key role in the pathogenesis of clinical and experimental asthma characterized by eosinophilic airway inflammation.