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CD4+CD25+ regulatory T (TR) cells play an important role in maintaining a balanced peripheral immune system.Recent studies have shown that TR cells may also play a key role in suppressing anti-tumor immune response. In order to investigate the tumor immune microenvironment and its influence on TR polarization, poorly immunogenic tumor cell line D5 (C57BL/6, H-2b), immunogenic tumor cell lines FBL3 (C57BL/6, H-2b) and H22 BALB/c, H-2d) were used to establish the syngeneic/allogeneic, poorly immunogenic/immunogenic mixed lymphocytes-tumor cell culture (MLTC). Our results revealed that the proportion of CD4+CD25+ T cells in MLTC of syngeneic primed splenocytes stimulated with D5 tumor cells was higher than that with H22 cells (0.43% vs 0.044%, and the similar results appeared in allogeneic splenocytes stimulated with D5 tumor cells (0.39% vs 0.04%).The splenocytes stimulated with supatant from syngeneic MLTC of D5 tumor cells demonstrated higher proportion of CD4+CD25+ cells than that from allogeneic MLTC of D5 tumor cells, and the splenocytes stimulated with supatant from syngeneic or allogeneic MLTC of H22 tumor cells generated lower proportion of CD4+CD25+ T cells than that of D5 tumor cells. The TGF-β1 and Th2-oriented cytokines (IL-4 and IL-10) were dominated in supatants of syngeneic MLTC of poorly immunogenic tumor cells. Our results provided useful information for studying the mechanisms underlying tumor immune surveillance as well as for the tumor immunotherapy.