Fabry disease is an X-linked lysosomal storage disorder that is caused by a deficiency of α-galactosidase A. The disease ultimately manifests as multiple organ dysfunctions owing to excessive accumulation of globotriaosylceramide (Gb3). Among the several complications of Fabry disease, ascending thoracic aortic aneurysm is relatively common, which is classically associated with connective tissue disorders characterized by abnormal defects or deficiencies in structural proteins such as collagen and elastin. Although an elevated Gb3 level is regarded as a prerequisite for the manifestations of Fabry disease, only this excess accumulation cannot explain the pathophysiology of these complications. Recently, an increased plasma level of lyso-Gb3 was suggested as a new biomarker in Fabry disease. Therefore, the aim of this study was to assess the effects of lyso-Gb3 on the pathogenesis of thoracic ascending aortic aneurysms in Fabry disease, with a particular focus on the responses related to aortic remodeling by fibroblasts. We found that lyso-Gb3 inhibited the growth of fibroblasts, as well as their differentiation into myofibroblasts, and collagen expression. Moreover, all of these compromised responses could be attributed to the effects of lyso-Gb3 on downregulation of KCa3.1 channel expression, and these impairments could be rescued when activating the KCa3.1 channel or increasing intracellular Ca(2+) concentration. This study provides new evidence that lyso-Gb3 inhibits the differentiation into myofibroblasts and collagen synthesis of fibroblasts owing to decreased Ca(2+) levels by KCa3.1 channel dysfunction. These findings suggest that the KCa3.1 channel can serve as a new target to attenuate and prevent development of ascending thoracic aortic aneurysm in Fabry disease.