Semaphorin 5A (SEMA5A) is an axonal regulator molecule, which belongs to the Semaphorin family of proteins. Previously, we identified SEMA5A as a putative marker for aggressive pancreatic tumors. However, the expression, localization and functional significance of SEMA5A in pancreatic tumors remain unclear. In our study, we hypothesized that SEMA5A expression modulates pancreatic tumor growth and metastasis. We analyzed the constitutive expression and localization of SEMA5A in patient pancreatic tumors (n = 33) and unmatched normal pancreatic (n = 8) tissues and human pancreatic cancer cell lines (n = 16) with different histopathological characteristics. We observed significantly higher expression of SEMA5A protein expression (p < 0.05) in human pancreatic tumor tissue samples compared to normal pancreatic tissues. Similarly, the pancreatic cancer cell lines with higher tumorigenic and metastatic potentials as xenografts in nude mice expressed higher levels of SEMA5A mRNA compared to those with lower tumorigenic and metastatic potentials. Furthermore, we examined the functional role of SEMA5A in pancreatic tumor growth and invasion. Ectopic expression of mouse full-length Sema5A in Panc1 (SEMA5A negative) cells significantly (p < 0.05) enhanced tumorigenesis, growth and metastasis in vivo as well as proliferation, invasiveness and homotypic aggregation in vitro. Together, these data demonstrate that the expression of SEMA5A in pancreatic cancer cells regulates tumorigenesis, growth, invasion and metastasis, and it also suggests a novel target for diagnosis and treatment of pancreatic cancer.