Background Adoptive cell transfer (ACT) immunotherapy has been used clinically for years to treat malignancies. Improving the killing efficiency of effector cells, such as tumor-specific cytotoxic T lymphocytes (CTLs), is an important component for enhancing the clinical response of cancer immunotherapy. Hence, we explored a novel method for preparing cancer-specific CTLs using naive T lymphocytes. Methods C57BL/6 mice bearing B16 melanoma tumors were pretreated with cyclophosphamide (CTX) by peritoneal injection. The immunosuppressive influence of CTX on tumor regression and the tumor microenvironment was assessed. Naive T cells and T cell pools were isolated via negative selection using immunomagnetic beads. The proliferative potential and cytokine production of different T cell subpopulations were evaluated in vitro. Tumor-specific CTLs derived from naive T cells (naive CD4+ T cells: naive CD8+ T cells=2:1) and pooled T cells were generated in vitro, respectively. B16 melanoma-bearing C57BL/6 mice were pretreated with CTX, followed by ACT immunotherapy using dendritic cell-induced CTLs. The homing abilities of the effector cells and interleukin-2 (IL-2), interferon-gamma, granzyme B, and perforin mRNA levels in tumor tissues were evaluated, and the change in tumor volume was measured. Results Mice receiving CTX peritoneal pretreatment injections did not display tumor regression compared with control mice. However, a significant downregulation of splenic Tregs and tumor growth factor-beta 1 (TGF-beta 1) and interleukin-10 (IL-10) serum levels was observed (P<0.05). Naive T cells showed a stronger proliferative capacity and elevated cytokine production than did pooled T cells (P<0.05). In addition, effector cells generated from naive T cells displayed more potent antitumor activity in vivo than those derived from pooled T cells (P<0.05). Conclusion Effector cells derived from the naive T cells possess a stronger proliferative potential, homing capacity, and enhanced cytokine production, which leads to a superior antitumor response.