Dendritic cells (DC) play pivotal roles in the induction and regulation of both innate and acquired immunity. DC express several cell‐surface immune inhibitory receptors. However, little is known about their potential immunoregulatory functions in the context of T‐cell activation. Here we report that murine gp49B, a member of the immunoglobulin superfamily, harboring immunoreceptor tyrosine‐based inhibitory motifs, is expressed on DC and downregulates cellular activity to prevent the excessive activation of T cells in vitro and in vivo. Bone marrow‐derived DC (BMDC) from newly generated gp49B‐deficient (gp49B^−/−) mice induced enhanced proliferation and IL‐2 release of antigen‐specific CD4⁺ and CD8⁺ T cells compared with BMDC from wild‐type mice, in a cell–cell contact manner. The enhanced proliferation by gp49B^−/− BMDC was also observed in allogeneic CD4⁺ and CD8⁺ T cells. Moreover, the transfer of allogeneic BALB/c splenocytes into C57BL/6 gp49B^−/− mice induced severe acute graft‐versus‐host disease with an augmented upregulation of CD86 on CD11c⁺ splenic gp49B^−/− DC, while transfer of C57BL/6 gp49B^−/− splenocytes into BALB/c mice did not, suggesting the exacerbation of the disease was due, at least in part, to augmented activation of recipient gp49B^−/− DC. These findings demonstrate a novel regulatory role of gp49B in the function of DC.