M2-like tumor-associated macrophages (TAMs) are the main immunosuppressive cells infiltrating the tumor microenvironment (TME), the activation of which is essential for cancer progression and resistance promotion to immunotherapy. However, the regulatory mechanisms underlying TAM activation have not been fully elucidated. Utilizing a CRISPR-Cas9-based genetically engineered mouse model, we discovered that USP1Lyz2 and WDR48Lyz2 mice exhibited decreased tumor formation and lung metastasis. Mechanistically, the USP1-WDR48 deubiquitinase complex regulated M2-TAM activation and infiltration in the TME by modulating DDX3X ubiquitination. Specifically, this complex interacted with the N-terminal RecA-like domain 1 of DDX3X, leading to K48-linked deubiquitination and stabilization of DDX3X. Then, DDX3X promoted the translation of signaling molecules Jak1 and Rac1 via its RNA helicase activity, activating the Jak1-Stat3/6 and Rac1-Akt pathways to drive M2-TAM activation. Furthermore, combined inhibition of the USP1/WDR48 and CD47/SIRPα signaling pathways showed synergistic antitumor effects in immunocompetent mice. Notably, USP1 protein expression in tumor stromal tissues independently predicts prognosis in breast cancer patients. These findings indicated the role of the USP1-WDR48 complex as a critical molecular switch controlling TAM activation, presenting novel and promising targets for breast cancer treatment.