Background <p>Gastric cancer (GC) continues to pose a significant global health burden, necessitating a thorough understanding of the molecular mechanisms driving its progression. This study aimed to investigate the regulatory role of SMAD3 in modulating Sestrin2 ubiquitination stability and its implications in GC cell behaviors.</p> Methods <p>Leveraging GC-related single-cell transcriptomic data, we employed advanced analytical approaches to unveil the relationship between SMAD3 and Sestrin2. Experimental validations involving RT-qPCR, Western blotting, and immunofluorescence analyses elucidated the impact of SMAD3 on Sestrin2 expression and subcellular localization. In vitro models of SMAD3 overexpression and knockdown were utilized to assess the functional consequences on GC cell proliferation, migration, invasion, and apoptosis. The interaction between SMAD3 and ubiquitination-related enzymes RNF167 and STAMBPL1 was investigated through immunoprecipitation assays.</p> Results <p>Our findings revealed a positive correlation between SMAD3, Sestrin2, and STAMBPL1 expression, indicating a regulatory network within GC cells. SMAD3 was shown to stabilize Sestrin2 protein levels by influencing the ubiquitination processes of STAMBPL1 and RNF167. Functional assays demonstrated the promotion of GC cell proliferation, migration, and invasion, along with decreased apoptosis, by the SMAD3-Sestrin2 axis. Moreover, SMAD3-mediated regulation of Sestrin2 stability was found to enhance GC metastasis.</p> Conclusion <p>The study underscores the critical role of SMAD3 in modulating Sestrin2 expression and stability, consequently impacting GC cell behaviors and metastatic potential. The SMAD3-Sestrin2 axis emerges as a promising therapeutic target for GC treatment.</p> Graphic abstract <p>Multimodal analysis reveals the molecular mechanism by which SMAD3 mediates the synergistic regulation of Sestrin2’s polyubiquitination levels through RNF167 and STAMBPL1 to induce GC.</p>