AbstractPancreatic cancer (PC) is one of the most malignant gastrointestinal tumors and the 5‐year survival is only 9%. The expression of miRNAs in serum has been proved to be related to tumorigenesis and development of cancers. The miRNA targets and gene targets were predicted in microRNA.org, miRDB, TargetScan, and RNAInter. The expression data of STK31 (Serine/Threonine Kinase 31) and miRNAs generated from PC samples was from TCGA and the relationship of expression of STK31 and miR‐543 was confirmed in PC samples from our center. Double luciferase reporter gene assay was used to demonstrate the direct binding between miR‐543 and STK31. The effect of expression level of miRNAs on survival time was assessed by Kaplan–Meier curves. The Go Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miR‐543‐related genes were performed. The results showed that miR‐543 had a statistically significant correlation with the expression of STK31 and contained the direct binding site with STK31. The expression level of miR‐543 may affect the survival of PC. The results of GO and KEGG pathway analysis showed that miR‐543 might play a key role in Insulin signaling pathway. MiR‐543 could be combined with STK31 and affect the expression of STK31. The expression of miR‐543 could also predict the survival of patients with PC, which suggested that miR‐543 might play an important role in PC. The GO and KEGG pathway analysis also displayed that miR‐543 was involved in several other pathways of pancreas.