Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with poor prognosis, and the interaction between activated pancreatic stellate cells (PSCs) and PDAC cells plays an important role in the development of PDAC. The aim of this study was to identify gene changes in BXPC-3 after cross-talk with PSCs and reveal their potential mechanisms. The gene expression profiling analysis of BXPC-3 was completed after co-culture with primary PSCs for 48 h. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed, and the differentially expressed genes (DEGs) were identified by Agilent GeneSpring GX software. In total, 3657 DEGs were identified in BXPC-3, including 1881 up-regulated genes and 1776 downregulated genes. GO analysis results showed that upregulated DEGs were significantly enriched in biological processes (BP), including peptide metabolic process, response to stress and electron transport chain; the downregulated DEGs were significantly enriched in biological processes, including signaling, multicellular organism development and anatomical structure development. KEGG pathway analysis revealed that 19 pathways were upregulated and 32 pathways were downregulated, and that upregulated DEGs were enriched in protein export and glutathione metabolism, while the downregulated DEGs were enriched in axon guidance and focal adhesion. The top 10 upregulated genes and the top 10 downregulated genes were identified. By constructing PPI network, we selected out 10 key genes (TP53, SRC, IL6, JUN, ISG15, CAD, STAT1, OAS3, OAS1, VIM) and significant pathways. The associated survival analysis was performed and the SRC, IL-6, ISG15, STAT1, OAS3, OAS1 and VIM were proved to be related to worse overall survival time of PDAC patients. In conclusion, the present study indicated that the identified DEGs promote our understanding of the molecular mechanisms underlying the interaction between pancreatic cancer cells and PSCs and might be used as molecular targets in the future to study the role of tumor microenvironment in the progression of PDAC.