Background and Aims Pancreatic cancer is characterized by high metastatic potential and invasiveness, and dismal prognosis. However, Mutations in oncogenes and changes in molecular regulatory relationships can affect the malignant biological behaviors of pancreatic cancer. The expression of ubiquitin/ISG15-conjugating enzyme E2L6 (UBE2L6) in pan-cancer and its biological function in pancreatic cancer remain unclear. Therefore, this study was performed to explore the effect of UBE2L6 on the proliferation, migration, and invasion abilities of pancreatic cancer and its underlying molecular mechanism through bioinformatic analysis and experimental verification.Methods The RNA-seq data of TCGA and GTEx were downloaded from UCSC Xena, and the differential expressions of UBE2L6 in pan-cancer were analyzed using the “limma” package of R (4.0.2). The differential expression of UBE2L6 in pancreatic cancer tissue and cell lines was verified by the Gene Expression Omnibus (GEO) datasets and qRT-PCR, respectively. In pancreatic cancer cells after transfection with siRNA targeting UBE2L6, the influences of UBE2L6 on the biological functions of pancreatic cells were analyzed by CCK-8 proliferation, colony formation, Transwell migration and invasion assays. The potential action mechanism of UBE2L6 were explored by the molecular correlation, protein interaction, and gene set enrichment analysis (GSEA). Then, the clinical data of pancreatic cancer were downloaded from the TCGA database to analyze the association of UBE2L6 expression with the clinicopathologic features and its clinical application value.Results Differential expression analysis suggested that UBE2L6 expression was up-regulated in pan-cancer as well as in pancreatic cancer tissue and a variety of pancreatic cancer cell lines such as BxPC-3 (t=33.82, P<0.000 1), PANC-1 (t=7.36, P=0.001 8), AsPC-1 (t=9.61, P=0.000 7), SW1990 (t=10.26, P=0.000 5), and MIA PaCa-2 (t=12.65, P=0.000 2). Cell function assays showed that UBE2L6 promoted the proliferation, migration, and invasion of PANC-1 and AsPC-1 cells. Spearman correlation analysis indicated that BCL2A1 protein had the highest correlation with UBE2L6 (r=0.442). The protein interaction analysis showed that it potentially interacted with HERC6, RPS27A, HERC5, UBA52, ISG15, UBA7, DDX58, UBC, UBB, and ARIH1. Meanwhile, GSEA enrichment analysis suggested that UBE2L6 was closely related to the tumor immune microenvironment. While clinicopathologic correlation analysis showed that the high expression of UBE2L6 was closely correlated with the histopathological grade of pancreatic cancer (χ²=6.966, P=0.031). Next, univariate and multivariate Cox regression analysis showed that age and lymph node metastasis were independent risk factors for pancreatic cancer patients (both P<0.05), and the expression of UBE2L6 was significantly correlated with the prognosis of pancreatic cancer patients (P<0.05), but it was not an independent prognostic factor (P>0.05). Furthermore, ROC curve and Kaplan-Meier survival analysis suggested that UBE2L6 had a high clinical diagnostic value for pancreatic cancer (AUC=0.970, 95% CI=0.950-0.989) and was correlated with the prognosis of pancreatic cancer patients (P<0.05).Conclusion UBE2L6 is highly expressed in pancreatic cancer, and it promotes the proliferation, migration, and invasion of pancreatic cancer cells. Its molecular mechanism may be related to cell apoptosis, ubiquitination, the MAPK signaling pathway, and tumor immune microenvironment. In addition, UBE2L6 is closely associated with the diagnosis and prognosis of pancreatic cancer.