Immunotherapy has achieved prominent clinical efficacy in combating cancer and has recently become a mainstream treatment strategy. However, achieving broad efficacy with a single modality is challenging, and the heterogeneity of the tumor microenvironment (TME) restricts the accuracy and effectiveness of immunotherapy strategies for tumors. Herein, a TME-responsive targeted nanoparticle to enhance antitumor immunity and reverse immune escape by codelivering interleukin-12 (IL-12) expressing gene and colony-stimulating factor-1 receptor (CSF-1R) inhibitor PLX3397 (PLX) is presented. The introduction of disulfide bonds and cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptides conferred reduction reactivity and tumor targeting to the nanoparticles, respectively. It is hypothesized that activating host immunity by the local expression of IL-12, while modulating the tumor-associated macrophages (TAM) function through blocking CSF-1/CSF-1R signaling, could constitute a feasible approach for cancer immunotherapy. The fabricated functional nanoparticle successfully ameliorated the TME by stimulating the proliferation and activation of T lymphocytes, promoting the repolarization of TAMs, reducing myeloid-derived suppressor cells (MDSCs), and promoting the maturation of dendritic cells (DC) as well as the secretion of antitumor cytokines, which efficiently suppressed tumor growth and metastasis. Finally, substantial changes in the TME were deciphered by single-cell analysis including infiltration of different cells, transcriptional states, secretory signaling and cell-cell communications. These findings provide a promising combinatorial immunotherapy strategy through immunomodulatory nanoparticles.