AMD3100 (plerixafor), a CXCR4 antagonist, has been demonstrated to suppress tumor growth and modulate intratumoral T-cell trafficking. However, the effect of AMD3100 on immunomodulation remains elusive. Here, we explored immunomodulation and antitumor efficacy of AMD3100 in combination with a previously developed mesothelin-targeted, immune-activating fusion protein, VIC-008, in two syngeneic, orthotopic models of malignant mesothelioma in immunocompetent mice. We showed that combination therapy significantly suppressed tumor growth and prolonged animal survival in two mouse models. Tumor control and survival benefit were associated with enhanced antitumor immunity. VIC-008 augmented mesothelin-specific CD8+ T-cell responses in the spleen and lymph nodes and facilitated intratumoral lymphocytic infiltration. However, VIC-008 treatment was associated with increased programmed cell death protein-1 (PD-1) expression on intratumoral CD8+ T cells, likely due to high CXCL12 in the tumor microenvironment. AMD3100 alone and in combination with VIC-008 modulated immunosuppression in tumors and the immune system through suppression of PD-1 expression on CD8+ T cells and conversion of regulatory T cells (Tregs) into CD4+CD25-Foxp3+IL2+CD40L+ helper-like cells. In mechanistic studies, we demonstrated that AMD3100-driven Treg reprogramming required T cell receptor (TCR) activation and was associated with loss of PTEN due to oxidative inactivation. The combination of VIC-008 augmentation of tumor-specific CD8+ T-cell responses with AMD3100 abrogation of immunosuppression conferred significant benefits for tumor control and animal survival. These data provide new mechanistic insight into AMD3100-mediated immunomodulation and highlight the enhanced antitumor effect of AMD3100 in combination with a tumor antigen-targeted therapy in mouse malignant mesothelioma, which could be clinically relevant to patients with this difficult-to-treat disease. Cancer Immunol Res; 6(5); 539-51. ©2018 AACR.