High mobility group box 1 protein (HMGB1) has been identified as a late proinflammatory cytokine and plays a key role in immune regulation. However, it is not yet clear whether HMGB1 can induce the activation and differentiation of dendritic cell (DC) subsets and subsequently modulate immune function of T cells. This study was performed to investigate the effect of HMGB1 on the differentiation of splenic DCs and its influence on T cell-mediated immunity in terms of DC subsets CD11c low CD45RB high DCs and CD11c high CD45RB low DCs in male BALB/c mice spleens in vitro.MACS microbeads were used to isolate splenic DCs, CD11c low CD45RB high DCs, CD11c high CD45RB low DCs and CD4 + T cells. The percentage of CD11c low CD45RB high DCs was significantly increased after treatment with HMGB1 compared to their counterparts (CD11c high CD45RB low DCs). It was found that unlike the gradually increasing interleukin (IL)-12 secretion of CD11c high CD45RB low DCs induced by HMGB1, CD11c low CD45RB high DCs showed a obvious dose-dependent response between IL-10 production and HMGB1 stimulation. In order to verify whether the alteration of CD4 + T cells was mainly associated with the differentiation of splenic DCs mediated by HMGB1 to CD11c low CD45RB high DCs, anti-IL-12 receptor (IL-12R) or anti-IL-10R monoclonal antibody was used to inhibit the effect of CD11c high CD45RB low DCs or CD11c low CD45RB high DCs in CD4 + T cells mixed lymphocyte reaction culture. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4 + T cells+CD11c high CD45RB low DCs or CD11c low CD45RB high DCs mixed lymphocyte reaction, the induction of these DCs on T cells was inhibited dramatically.These data demonstrated that HMGB1 might induce the differentiation of splenic DCs to CD11c low CD45RB high DCs followed by shifting of Th1 to Th2 with enhancement of T lymphocyte immune function in vitro. Also, the effect of HMGB1 on T cell differentiation to Th2 was not associated with the inhibition of IL-12 production in CD11c high CD45RB low DCs.