In this study, we systematically revealed the expression characteristics, clinical relevance, and potential molecular mechanisms of the ubiquitin-specific processing protease 35 (USP35) in 33 cancers for the first time by integrating pan-cancer data from TCGA, GTEx, and other databases. Bioinformatics analysis showed that USP35 was significantly highly expressed in nine cancers, including gastric cancer (GC) and thyroid cancer, and its expression level was closely related to the tumor stage, metastasis, and a poor prognosis. Diagnostic value analysis showed that the area under the curve (AUC) value of USP35 exceeded 0.7 for four cancers, including rectal adenocarcinoma, suggesting its potential as a diagnostic marker. Mechanistic studies revealed that USP35 may affect tumor progression by regulating myogenesis, the epithelial-mesenchymal transition (EMT), and other pathways, and is significantly associated with apoptosis, cell cycles, and other activities. Experimental validation partially confirmed that USP35 is strongly associated with cancer-associated fibroblasts in GC. Knockdown of USP35 inhibited GC cell migration/invasion, down-regulated vimentin, and blocked energy metabolism reprogramming through the inhibition of glycolysis. In combination with USP35 knockdown, 2-DG further exacerbated the metabolic inhibitory effects, and the reversal of the EMT was even more significant. USP35 enhanced the tumorigenic capacity and the EMT of GC cells in vivo. This study demonstrates that USP35 may promote GC progression through metabolic reprogramming, exhibiting potential as a diagnostic and prognostic tumor marker.