Pancreatic ductal carcinoma (PDAC) is a common malignant tumor of the digestive system. GATA4 is one of the transcriptional regulatory factors, which regulates the development of endoderm-derived organs, including heart and gut. GATA4 may act as a putative tumor suppressor gene. However, the role of GATA4 in pancreatic carcinogenesis is not yet clarified. This study showed that GATA4 was highly expressed in pancreatic cancer tissues, and its expression level was positively related to the grade of pathological differentiation, suggesting that it may contribute to the progression of pancreatic neoplasia. Ectopic expression of GATA4 gene reduced cell viability and interference of GATA4 expression significantly increased the colony formation ability of pancreatic cancer cells. Furthermore, GATA4 inhibited tumor growth in xenograft mice. Agilent expression microarray profiling analysis indicated that the genes with significant levels of differential expression in GATA4 over-expressing cells were enriched in the cell differentiation process. Analysis of KEGG signaling pathway demonstrated that the regulated genes were partially enriched in MAPK and JAK-STAT signaling pathways. Re-expression of GATA4 up-regulated P53 gene expression. Our data indicate that GATA4 gene might play a role in cell proliferation and differentiation during the progression of pancreatic cancer.