Background Mepolizumab, a humanized antibody targeting interleukin-5, decreases the number of blood eosinophils and the frequency of exacerbation of severe asthma. Galectin-10 is a protein within the cytoplasm of eosinophils and constitutes Charcot-Leyden crystals, which promotes key features of asthma. However, the relationship between time kinetics and clinical response of eosinophil-derived molecules such as galectin-10 or eosinophil cationic protein (ECP) has not been precisely investigated. Objective This study aimed to clarify the precise time course of the levels of serum galectin-10 and ECP after mepolizumab treatment and to analyze the relationship between the levels of eosinophil-derived molecules and the clinical background or response to mepolizumab treatment. Methods This multicenter, prospective open-label study recruited 20 patients with severe eosinophilic asthma. Mepolizumab was administered every 4 weeks for 32 weeks and the levels of various biomarkers were serially analyzed. Results The serum galectin-10 and ECP significantly and rapidly decreased 4 weeks after initial administration of mepolizumab. In contrast, basophil count, fractional exhaled nitric oxide, and the serum total IgE level were unchanged during treatment. Asthma Control Questionnaire-5, Asthma Health Questionnaire-33, and Lund-Mackay scores significantly improved after mepolizumab treatment. Both high ECP and eosinophil count related to better response in forced expiratory volume in 1 second (FEV1) and measurable ECP level at 4 weeks after administration of mepolizumab related to the further improvement in FEV1 toward week 32. No significant difference in improvement in FEV1 was observed in galectin-10 high group. The level of ECP at baseline was significantly related to the higher prevalence of nasal polyp and Lund-Mackay score. Conclusion This study was the first to show that the levels of serum galectin-10 decreases after initial administration of mepolizumab. The significant relationship between serum ECP and better response in FEV1 suggested the potential role of serum ECP as a predictive biomarker for the efficacy of mepolizumab (UMIN000030466).