Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3(+) CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3(+) CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3(+) CD4 T cells. The majority of tumor-derived Tim-3(+) CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3(-) CD4 T cell counterparts. In contrast, most Tim-3(+) CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3(+) CD4 T cells, but not tumor-derived Tim-3(-) CD4 T cells, significantly suppressed the proliferation of autologous CD8(+) T cells in vitro. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3(+)Foxp3(+)CD4(+) cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.