Trastuzumab, a humanized antibody targeting human epidermal growth factor receptor 2 (HER2), exhibits remarkable therapeutic efficacy against HER2-positive gastric cancer. Acquired resistance to trastuzumab remains a barrier to patient survival and the mechanisms underlying this are still not well understood. The normal epithelial cell-specific-1 (NES1) gene, also named as KLK10, is recognized as a potential therapeutic target for reversing trastuzumab resistance. The aim of this study was to explore the potential role of KLK10 in trastuzumab resistance (TR) gastric cancer cells. We found that KLK10 was significantly upregulated in trastuzumab-resistant cell lines, SGC7901-TR and BGC-823-TR. In addition, down regulation of KLK10 reversed the resistance in trastuzumab resistant cells. Overexpression of KLK10 induced trastuzumab resistance, and activated the PI3K/AKT signaling pathway, while downregulation of KLK10 presented the opposite effects. Moreover, when the PI3K/AKT signaling pathway was inhibited, the effect of KLK10 on resistance was diminished. Furthermore, combination of trastuzumab and PI3K/AKT inhibitor XL147 effectively inhibited tumor growth in KLK10-overexpressing xenografts. Taken together, our findings show that KLK10 promotes trastuzumab resistance, at least in part, through the PI3K/AKT signaling pathway, suggesting that KLK10 is a potentially target to overcome trastuzumab resistance, and the combination might overcome trastuzumab resistance in KLK10-overexpressed gastric cancer patients.