Gallbladder cancer, the most prevalent malignant neoplasm of the biliary tract, has garnered significant attention due to its dismal prognosis and high degree of malignancy. Identifying key regulatory genes is crucial for the development of effective therapeutic strategies. The differential gene expression in biliary tract malignancies was identified using the Gene Expression Omnibus (GEO) database. Subsequently, the interactions among these differentially expressed genes were analyzed employing the STRING database, and the resultant regulatory network was visualized using Cytoscape software. Utilizing the Cytoscape plugin CytoHubba, the core genes within the network were identified, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Ultimately, the overexpression of THBS1 in the gallbladder cancer cell line (NOZ) was achieved through lentiviral transfection and both in vivo and in vitro experiments were conducted to evaluate its effects. We found that thrombospondin-1 (THBS1) was the core gene of gallbladder cancer and its expression was low in gallbladder cancer. Experimental data, both in vivo and in vitro, indicate that the up-regulation of THBS1 exerts an inhibitory effect on the proliferation, migration, and invasion of gallbladder cancer cells. Furthermore, it facilitates the process of apoptosis and suppresses tumor growth and angiogenesis. The expression of THBS1 is low in gallbladder cancer. Up-regulation of THBS1 can effectively inhibit the occurrence and development of gallbladder cancer and can be used as a biomarker for the diagnosis and treatment of gallbladder cancer.