Background <p>UPK1B has been implicated in various cancers; however, its mechanism of action in gastric cancer remains elusive.</p> Methods <p>We utilized transcriptional data and clinical information, and mutation profiles from The Cancer Genome Atlas (TCGA) database to analyze UPK1B's expression and clinical relevance. Biological enrichment, immune microenvironment characterization, and drug sensitivity analyses were conducted. Functional assays, including proliferation, migration, invasion, and in vivo metastasis models, were used to validate UPK1B's role in gastric cancer.</p> Results <p>UPK1B was significantly upregulated in gastric cancer and correlated with worse clinical outcomes, including advanced stages and reduced survival rates. Biological enrichment analysis revealed its involvement in cancer-related pathways such as DNA replication and immune regulation. UPK1B was negatively correlated with NK cells and M1 macrophages, indicating its role in immune evasion. Functional experiments demonstrated that knockdown of UPK1B significantly suppressed gastric cancer cell proliferation, invasion, and migration in vitro and reduced pulmonary metastases in vivo. Drug sensitivity analysis suggested that high UPK1B expression was associated with increased sensitivity to lapatinib and resistance to cisplatin.</p> Conclusions <p>UPK1B promotes tumor progression and modulates the immune microenvironment in gastric cancer, making it a potential therapeutic target for future research and clinical applications.</p>