Objective: To investigate the expression characteristics of protein tyrosine phosphatase non-receptor type 6(PTPN6)in gastric cancer tissues, evaluate its correlation with natural killer (NK) cell infiltration and activation, and further elucidate the regulatory role and molecular mechanisms of PTPN6 in malignant phenotypes such as gastric cancer cell proliferation and that in NK cell cytotoxic activity. Methods: Based on the Asian Cancer Research Group(ACRG)gastric cancer cohort and integrated single-cell data, the expression level of PTPN6 in gastric cancer tissues and its correlation with NK cell infiltration and activation levels were analyzed. CCK-8, EdU, colony formation, and Transwell assays were used to evaluate the effects of silencing or overexpressing PTPN6 on the proliferation, migration, and invasion of MKN1 and AGS cells. Flow cytometry was employed to detect apoptosis levels and NK cell-mediated killing efficiency. qRT-PCR was used to measure the expression levels of human leukocyte antigen-class Ⅰ(HLA-Ⅰ) molecules. Whole-exome sequencing was applied to assess the genomic similarity between gastric cancer organoids and primary tissues, and a co-culture model of gastric cancer organoids and NK cells was used to evaluate NK cell killing efficiency. A PTPN6-overexpressing YTN16 gastric cancer cell line was constructed and subcutaneously inoculated into NCG and C57BL/6 mice, followed by flow cytometric analysis of tumor-infiltrating NK cells. Transcriptome sequencing was performed to identify potential downstream molecules and signaling pathways regulated by PTPN6. Results: PTPN6 expression was significantly upregulated in gastric cancer tissues and negatively correlated with NK cell infiltration and activation levels. Knockdown of PTPN6 inhibited gastric cancer cell proliferation, enhanced NK cell killing sensitivity, and downregulated HLA-I molecule expression. In contrast, PTPN6 overexpression promoted malignant phenotypes of gastric cancer cells, reduced NK cell killing activity, and upregulated HLA-I molecule expression. In vivo mouse xenograft experiments showed that PTPN6 overexpression accelerated tumor growth in C57BL/6 mice and reduced the proportion of NK cell infiltration in tumor tissues. Transcriptome sequencing analysis indicated that PTPN6 overexpression activated signaling pathways such as PI3K/AKT, MYC, and NF-κB. Conclusion: PTPN6 is highly expressed in gastric cancer and promotes tumor cell proliferation and invasion by activating signaling pathways such as PI3K/AKT. Its expression is negatively correlated with NK cell infiltration and activation levels, and it mediates immune escape by regulating HLA-I molecule expression in gastric cancer cells. This study provides a new potential target and theoretical basis for immunotherapy in gastric cancer.