Many homeobox (HOX) genes have been shown to be related to cancer progression. HOXB5, a member of the HOX genes, is overexpressed in retinoblastoma cancer and positively regulates the breast cancer cell proliferation as well as invasion. However, the role and underlying mechanism of HOXB5 in pancreatic cancer cells are still unclear. HOXB5 expression was measured in four pancreatic cancer cell lines, including PANC-1, ASPC-1, MIA-PaCa-2, and CFPAC-1. PANC-1 and ASPC-1 cells were selected for cell transfection experiments. Cell proliferation, migration, and invasion were measured by Cell Counting Kit-8 (CCK-8) assay, wound healing assay, and transwell assay. Expressions of epithelial-to-mesenchymal transition (EMT) markers were determined by western blotting. Immunofluorescence staining and cellular morphology were used to confirm the effect of HOXB5 dysregulation on pancreatic cancer cells. We found that HOXB5 was markedly expressed in pancreatic cancer cell lines. HOXB5 overexpression contributed to proliferation, migration, and invasion in ASPC-1 cells, whereas HOXB5 knockdown decreased proliferation, migration, and invasion of PANC-1 cells. Western blotting confirmed that overexpression of HOXB5 promoted the EMT process. Conversely, knockdown of HOXB5 alleviated EMT. Furthermore, knockdown of HOXB5 suppressed proliferation, migration, and invasion of pancreatic cancer cells via the Glycogen synthase kinase 3β (GSK3β)/β-catenin pathway. Our study demonstrates that HOXB5 is a tumor promoter in pancreatic cancer, and the GSK3β/β-catenin pathway is important in HOXB5-induced proliferation, migration, and invasion in pancreatic cancer cells.