Purpose: Referring to global cancer statistics, the incidence of gastric cancer (GC) was ranked sixth; however, detailed mechanisms underlying its development were not thoroughly investigated. Previous studies have reported that inhibition of ubiquitin-specific peptidase 8 (USP8) induced degradation of several receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), embryonic stem cells (ESCs), etc. Nevertheless, the regulation of HER-2 by USP8 and the molecular mechanisms controlling their role in the pathogenesis of GC remain unknown. Patients and Methods: A total of 69 patients with histologically confirmed GC were recruited to satisfy the purpose of this study. Initially, tumor samples and GC cell lines were used to detect USP8 and HER-2 levels. Next, MTT and colony formation assays were applied to analyze cell proliferation capability. Cell migration and invasion ability were examined by transwell assays. To examine related mRNA and protein expressions, Western blot assays and quantitative real-time PCR (qRT-PCR) were performed. Immunofluorescence was used to detect the effect of USP8 inhibitor on GC cells. Finally, in vivo experiments were used to examine the effect of USP8 inhibitor. Results: Patients with USP8 high-expression tumors have shown worse overall survival while opposite results found in patients with low USP8 expressions. Regarding disease prognosis, patients with low expression of USP8 and HER-2 were performed better prognosis, whereas those with overexpression of USP8 and HER-2 shown poor prognosis. USP8 inhibitor significantly inhibited HER-2 positive cell NCI-N87 proliferation and metastasis. In addition, USP8 stabilizes HER-2, preventing it from ubiquitin proteasome-mediated degradation. In vivo studies confirmed that the USP8 inhibitor inhibited HER-2 positive cell NCI-N87 tumor growth. However, it did not affect the HER2-negative cell MGC-803. Careful investigation unraveled that the USP8 inhibitor significantly inhibited NCI-N87 cell proliferation and metastasis via phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/AKT) pathway. Conclusion: The USP8 inhibited HER-2 positive GC cell proliferation and migration in vivo and in vitro and probably served as a novel potential therapeutic biomarker for HER-2 positive GC.