<p>Resistance to anti-PD-1 therapy remains a significant challenge in gastric cancer (GC) treatment. Here, we revealed that the USP14-IMP2-CXCL2 axis in tumor-associated macrophages (TAMs) drove resistance by recruiting myeloid-derived suppressor cells (MDSCs). Endoscopic biopsy samples were obtained from patients with inoperable GC who were candidates for anti-PD-1 therapy. Single-cell RNA sequencing (scRNA-seq) analysis showed a higher prevalence of USP14⁺ TAMs in therapy-resistant cases, where USP14 was linked to the immunosuppressive phenotype of TAMs. Clinically, GC samples with elevated USP14⁺ TAM infiltration exhibited decreased CD8⁺ T cell presence and increased MDSC infiltration. In vivo experiments further confirmed that USP14⁺ TAMs facilitated resistance to anti-PD-1 therapy in GC, reduced the infiltration of CD8⁺ T cells, and significantly increased the infiltration of MDSCs. In particular, USP14⁺ TAMs markedly enhanced the recruitment of MDSCs into the GC microenvironment through the secretion of CXCL2. Mechanistically, USP14 stabilized the m6A reader IMP2 through deubiquitination, thus enhancing CXCL2 expression and secretion. Conversely, the E3 ligase RNF40 facilitated IMP2 degradation via increasing its ubiquitination, with USP14 and RNF40 dynamically balancing IMP2’s protein abundance. Furthermore, animal experiments demonstrated that targeted intervention of USP14 markedly enhanced the sensitivity of GC to anti-PD-1 therapy. This study provided a comprehensive exploration of USP14’s oncogenic roles in TAMs, suggesting a novel strategy to enhance the efficacy of anti-PD-1 therapy by inhibiting the USP14/IMP2/CXCL2 signaling axis in GC.</p><p></p>