The roles of TGF-β and the interaction between TGF-β and EGFR signaling are critical in Sertoli cell, though the knowledge about them is limited. RT-PCR was used to characterize the status of TGF-β signaling in clinical testicular specimens with complete Sertoli cell-only syndrome (SCOS). The mouse Sertoli cell TM4 was used to investigate the interaction between TGF-β and EGFR signaling by using mitogenic assay, luciferase assay, and western blot, while TM3 (mouse leydig cell), 3T3 (mouse embryo fibroblasts), and B82 (mouse lung fibroblasts) were selected as control. The RT-PCR assay indicated that the expression levels of TβRII and Smad2 in SCOS testes were upregulated compared to that in the normal controls. In the in vitro experiment, the TGF-β1 downregulated cellular proliferation of TM3 and B82 cell (P < 0.05), but it did not changed the proliferation of TM4 and 3T3 cells (P > 0.05). On contrast, TGF-β1 only increased the TGF response elements p3TP-lux activity significantly (P < 0.05) in Sertoli cell TM4. Also, the Western blot assay shows an obvious increase of Smad2 in TM4, 3T3, and TM3 cells after TGF-β1 treatment while the EGFR expression level was significantly increased in TM4 cells only. In conclusion, the TGF-β pathway and the cross-link between TGF-β and EGFR signaling may play an important role on the dysfunction of Sertoli cells which induce germ stem cells' disappearance in SCOS.