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Triple-negative breast cancer (TNBC) is an aggressive subtype lacking effective targeted therapies. A multitarget gene therapy was developed and validated to overcome molecular heterogeneity and compensatory survival signaling in TNBC. A codon-optimized human transcriptional intermediary factor 1 gamma (opti-hTIF1γ) gene therapy was evaluated in ex vivo-cultured patient biopsy tissues and in orthotopic and mammary intraductal mouse models, and the underlying mechanisms were investigated using pathway and immune-functional analyses. Ex vivo findings were further validated using patient-derived cells and complementary mechanistic studies, including ubiquitination assays, chromatin immunoprecipitation, and functional macrophage coculture analyses, to define the molecular basis of TIF1γ-mediated antitumor activity. Transduction of opti-hTIF1γ to ex vivo-cultured biopsy tissues from TNBC patients suppressed epithelial-to-mesenchymal transition and proliferation while inducing apoptosis. In orthotopic and mammary intraductal mouse models, opti-hTIF1γ effectively suppressed tumor growth and lung metastasis. Mechanistically, opti-hTIF1γ inhibits β-catenin via ubiquitination-dependent degradation and inhibits the SMAD-dependent TGFβ pathway by binding to SMAD2/3. In parallel, it suppresses the SMAD-independent TGFβ pathway via ubiquitination and caspase-3-associated degradation of STAT3, leading to the inhibition of TAK1. Furthermore, opti-hTIF1γ downregulates STAT3-dependent immune modulators such as CD47 and CXCL5 in TNBC, enhancing macrophage phagocytosis. These findings position opti-hTIF1γ as a promising multitarget gene therapeutic strategy for TNBC through concurrent suppression of tumorigenic signaling and reprogramming of the immune landscape.