Anosmin-1 (ANOS1) is an extracellular matrix (ECM)-related glycoprotein that is highly expressed in a variety of tumors. However, the specific role and mechanism of ANOS1 in advanced gastric cancer (GC) and its correlation with epithelial-mesenchymal transition (EMT) are not well understood, despite ongoing research into the expression of ANOS1 and its impact on clinical outcomes. The ANOS1 expression and clinical data were acquired from The Cancer Genome Atlas (TCGA) database to analyze the differential expression of ANOS1 and its prognostic impact in advanced gastric cancer. The expression of ANOS1 and E-cadherin was detected using immunohistochemical (IHC) techniques in 99 cases of advanced gastric cancer (GC) tissues and their adjacent normal tissues. Subsequently, the correlation of ANOS1 with clinicopathological characteristics was performed to discuss using chi-square test. The prognostic value of ANOS1 expression was assessed using Kaplan-Meier survival analysis and Cox regression. The Spearman's study was conducted to explore the relationship between ANOS1 expression, immunity, and EMT-related genes. Gene set enrichment analysis (GSEA) was utilized to determine the functional enrichment of ANOS1 in gastric cancer and the "pRRophetic"R package was utilized to determine the relationship between ANOS1 expression and drug sensitivity. TCGA data analysis has shown that ANOS1 was significantly overexpressed in advanced GC and high ANOS1 expression had a poor prognosis. The IHC analysis showed elevated ANOS1 levels in advanced GC tissues compared to the adjacent tissues. High ANOS1 expression was correlated with tumor infiltration, lymph node metastasis, TNM stage, and vascular invasion in advanced GC, indicating its role in tumor invasiveness and metastasis. Furthermore, research indicated that the expression of ANOS1 was inversely associated with E-cadherin levels in advanced GC. Kaplan-Meier analysis demonstrated that patients with high ANOS1 expression had poorer overall survival than those with low expression. Furthermore, ANOS1 was positively associated with EMT-related genes and correlated with 22 types of immune cell infiltration and 7 immune checkpoints. The patients with high expression levels of ANOS1 demonstrated sensitivity to 5-fluorouracil, dasatinib, and docetaxel. GSEA analysis revealed significant enrichment of ANOS1 in multiple oncogenic pathways, particularly extracellular matrix (ECM) receptor interactions, focal adhesion, hematopoietic cell lineage, chemokine signaling pathways, pathways in cancer, and transforming growth factor-β signaling pathway. ANOS1 may impact gastric cancer progression by regulating EMT, suggesting its dual utility as both a prognostic biomarker and a novel therapeutic target for GC intervention.