High mobility group box 1 protein (HMGB1) was recently discovered to be a critical late-acting cytokine and innate immune-modulating factor in sepsis, but the potential role and mechanism of HMGB1 in adaptive immunity remains elusive. The present study demonstrated that HMGB1 had a dual influence on immune function of CD4⁺ T lymphocytes. Low dose of HMGB1 had no effect on the proliferation activity of CD4⁺ T lymphocytes, but the Th1 cytokines production was increased. In contrast, treatment with high amount of HMGB1 suppressed the proliferative response and induced Th2 polarization of CD4⁺ T lymphocytes. We found that the expression of mitofusin-2 (Mfn2; also named hyperplasia suppressor gene), a member of the mitofusin family, was decreased in CD4⁺ T lymphocytes when stimulated with high dose of HMGB1. Up-regulation of Mfn2 attenuated the suppressive effect of HMGB1 on CD4⁺ T lymphocytes, which was associated with profound elevation of intracellular calcium concentration ([Ca²⁺]_i) and nuclear factor of activated T cells (NFAT) activity. These results indicate that HMGB1 have a direct role on adaptive immunity, and the decrease of Mfn2 expression may be a major cause of HMGB1-mediated immune dysfunction and Ca²⁺-NFAT signaling defect of CD4⁺ T lymphocytes.