An imbalance in Th17 cells and Tregs may be an important cause of the pathogenesis of thymoma with myasthenia gravis (MG). In this study, 30 patients with simple thymoma and 30 patients with thymoma with MG were analyzed. Flow cytometry analysis of Th17 and Tregs in peripheral blood revealed that the percentages of Th17 in thymoma were lower than those in thymoma with MG, while the percentages of Tregs were higher than those in simple thymoma. Serum cytokine ELISA assays showed that IL-6 levels in simple thymoma were lower than those in MG patients. Further, Th17 and Tregs levels were detected by immunohistochemical double staining of thymoma tissue; the number of positive Th17 cells in thymoma with MG was higher than that in simple thymoma, while positive Tregs showed the opposite results. RORγt protein and mRNA expression in thymoma with MG were both higher than those in simple thymoma. FOXP3 protein and mRNA expression in the thymoma with MG group were lower than those in simple thymoma. The results of coculture of thymoma cells and CD4+ T cells showed that thymoma cells could promote the differentiation of Th17 cells and inhibit the Tregs. Overall, Th17 cells and related transcription factors and cytokines in thymoma with MG patients were higher than those in thymoma patients, whereas, Tregs showed the opposite results, the mechanism may be that thymoma can secrete IL6 and IL21. These findings indicated that imbalances in Th17/Tregs and RORγt/FOXP3 may account for the pathogeny of thymoma with MG.