Thymus exerts a prominent role in the establishment os central T-cell tolerance, as well as in the development of self major histocompatibility complex (MHC)-restricted T lymphocytes. Like others autoimmune diseases, type 1 diabetes emergence implies central or peripheric self tolerance breakdown. Environmental factors, especially enterovirus infections, are supposed to be involved in diabetes pathophysiology. Epidemiological studies have highlighted a frequent association between enterovirus Coxsackievirus B4 (CVB4) and type 1 diabetes. The aim of our work was to study whether a thymus infection by CVB4 could induce modifications of thymic function. In primary cultures of thymic epithelial cells (TEC), we detected viral proteins, positive- and negative- strand RNA, and infectious virus in the supernatants, meaning that TEC cultures were susceptible to CVB4 infection and that CVB4 induced a persistent infection in those cells. CVB4 also modulated TEC proliferation and cytokine, such as IL-6, GM-CSF and LIF secretions. Studies using fetal organ thymus culture (FTOC) showed that CVB4 induced a marked and progressive thymocytes depletion, in particular double positive (DP) and CD4+ cells. CVB4 replicated in those subpopulations, indeed positive- and negative-atrand RNA were detected. CVB4 also upregulated MHC class I expression on DP thymocytes. The upregulation of MHC expression required viral infection in DP cells. IL-6 and GM-CSF secretions were also involved in this phenomenom, but IFN-alpha was shown not to be involved. Taken together, our results showed the susceptibility of the human thymus to CVB4 infection, and an important thymic dysfuntion due to this infection. Our work is a novel approach in the understanding of the mechanisms of CVB4-induced type 1 diabetes.