It is becoming increasingly evident that the multifunctionality of effector cells at the single-cell level is an important factor to predict the quality of T-cell response in immunological protection. The significance of the multifunctionality of T cells in anti-tumor immunity, however, remains unclear. Here, we assessed the IFN-gamma and TNF-alpha production and CD107a mobilization in adoptively transferred tumor-antigen-specific CD8(+) T cells at the single-cell level. Tumor growth of the murine fibrosarcoma CMS5 was found to limit the induction of multifunctionality in the transferred cells. These cells exhibited insufficient acquisition of the CD25(high)GITR(high)CD62L(low) phenotype and reduced infiltration in tumor. Depletion of Treg facilitated the induction of high multifunctionality of the transferred cells even in the hosts with progressing tumor, leading to enhanced tumor regression. The multifunctionality of the transferred cells correlated with in vivo CTL activity, and T cells with high multifunctionality harvested from hosts with successful therapy induced tumor regression when re-transferred into the tumor-bearing hosts. These data suggest that the appearance of multifunctional CD8(+) effector T cells in vivo is a critical determinant of the success of anti-tumor immunotherapy and Treg play an important role in the mechanism inhibiting the induction of multifunctionality in effector cells.