A common feature seen in acute infections is a severe atrophy of the thymus. This occurs in the murine model of acute Chagas disease. Moreover, in thymuses from Trypanosoma cruzi acutely infected mice, thymocytes exhibit an increase in the density of fibronectin and laminin integrin-type receptors, with an increase in migratory response ex vivo. Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation. To date, the consequences of molecular changes promoted by parasite infection upon thymus have not been elucidated. Considering the importance of microRNA for gene expression regulation, 85 microRNAs (mRNAs) were analyzed in TEC from T. cruzi acutely infected mice. The infection significantly modulated 29 miRNAs and modulation of 9 was also dependent whether TEC sorted out from the thymus exhibited cortical or medullary phenotype. In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death. Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.