Our previous work showed that mice immunized with attenuated activated syngeneic T cells (aTCV) led to damping Treg function which resulted in enhancing anti-tumor immunity. It is well known that DC plays a very important role in controlling Th cell differentiation; whether DC involves Treg attenuation in immunized mice remained unknown. In this study, we provided evidence that increased mature DC (mDC) after immunization with aTCV skewed Th17 differentiation, which resulted in inhibition of Treg differentiation through IL-6 signaling pathway. In the present study, we found that the frequency of mDCs increased dramatically in the immunized mice accompanied by lower Treg cells compared to the controls. Moreover, both DCs and serum derived from the immunized mice suppressed Treg differentiation in vitro, respectively. mDCs generated from bone marrow precursor cells in vitro strongly inhibited Treg development and simultaneously drove Th17 differentiation with elevated IL-6 production. However, PD-L1, a potent Treg inducer did not show effect on Treg down-regulation. Assay with transwell systems showed that cell-cell contact was necessary for IL-6 production to a threshold to activate Th17 transcriptional factor RORγt and to inhibit Treg counterpart Foxp3. Our results implicate up-regulated Th17 development might be one of mechanisms of enhancing anti-tumor immunity induced by immunization with aTCV, which provide a novel insight in numerous mechanisms responsible for anti-tumor immunity.