Background and Aims Pancreatic cancer is an invasive disease with a late diagnosis and poor prognosis. The studies on the molecular mechanism of pancreatic cancer are of great significance in improving the prognosis of patients with pancreatic cancer. Annexin A5 (ANXA5) is closely related to the occurrence and development of human tumors, but the association of ANXA5 with the prognosis and its mechanism are not very clear. Therefore, this study was conducted to investigate the relationship between the expression of ANXA5 and the prognosis of pancreatic cancer and its action mechanism by bioinformatics analysis combined experimental verification.Methods The transcriptome and clinical data of pancreatic cancer were downloaded from TCGA and GEO databases (GSE15471, GSE16515, GSE21501). The expression of ANXA5 gene in pancreatic cancer tissue and adjacent normal tissue in GEO database was analyzed by R packet, and the expression of ANXA5 gene in pancreatic cancer tissue in TCGA database and normal tissue in GTEx database was analyzed by GEPIA online website. The effect of ANXA5 expression on the overall survival time of patients with pancreatic cancer was analyzed by Kaplan-Meier method, and the risk factors of pancreatic cancer were determined by univariate and multivariate Cox analysis. The possible signal pathway of ANXA5 in pancreatic cancer and its correlation were analyzed using GSEA. Finally, immunohistochemical method was used to detect the expression of ANXA5 in 49 cases of pancreatic cancer and adjacent tissues, and its relationship with prognosis and clinicopathologic features of pancreatic cancer was analyzed.Results The results of database analysis showed that ANXA5 expressions were significantly increased in pancreatic cancer tissues in GSE15471 and GSE16515 data sets as well as in TCGA database (all P<0.05); the overall survival time of patients with high expression of ANXA5 was significantly shorter than that of patients with its low expression in either GSE21501 data set or TCGA database (both P<0.05); the expression of ANXA5 was an independent risk factor for the prognosis of pancreatic cancer patients in TCGA (HR=1.819, 95% CI=1.058-3.126, P=0.03); ANXA5 gene was highly correlated with both TGF-β pathway and epithelial-mesenchymal transition (EMT) in pancreatic cancer. The results of analysis of the pancreatic cancer tissue samples showed that the expression of ANXA5 in pancreatic cancer was significantly higher than that in adjacent tissue (P<0.001), and the high expression of this gene predicted a poor prognosis (P=0.008 2); the expression of ANXA5 was an independent risk factor for the prognosis of pancreatic cancer patients in TCGA (HR=3.06, 95% CI=1.046-8.952, P=0.041); the expression of ANXA5 was significantly associated with clinical stage (P=0.000 94) and lymph node metastasis (P<0.001).Conclusion The expression of ANXA5 is increased in pancreatic cancer, and its high expression is a risk factor for the prognosis of patients with pancreatic cancer. The mechanism may be associated with its promoting the development of pancreatic cancer through TGF-β pathway and EMT process.