Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7, and KGF receptor (KGFR) play important roles in the growth of epithelial cells and are overexpressed in a variety of malignant epithelial tumors, including pancreatic ductal adenocarcinoma (PDAC). We previously reported that co-expression of KGF and KGFR in PDAC is associated with venous invasion, enhanced vascular endothelial growth factor A expression and poor prognosis. Matrix metalloproteinase-9 (MMP-9) is known to participate in the degradation of type IV collagen, which is a primary component of extracellular matrices in the vascular basement membrane. In the present study, we examined the expression and roles of KGF, KGFR and MMP-9 in human PDAC cell lines and tissues. Quantitative real-time polymerase chain reaction analysis demonstrated the expression of MMP-9 mRNA in all eight PDAC cell lines. KGF, KGFR and MMP-9 were, respectively, expressed in 27 (43%), 23 (37%) and 35 (56%) of 63 patients. Each expression of KGF, KGFR or MMP-9 correlated positively with venous invasion. Furthermore, expression of KGF or MMP-9 correlated positively with liver metastasis. KGF-positive cases exhibited shorter survival than KGF-negative cases, while KGFR and MMP-9 expression were unrelated to prognosis. Administration of recombinant human KGF increased MMP-9 expression in PDAC cells, while transient transfection with short hairpin RNAs targeting KGF transcripts reduced MMP-9 expression in PDAC cells. Moreover, recombinant human KGF significantly enhanced migration and invasion of PDAC cells. These findings suggest that KGF and KGFR promote venous invasion via MMP-9 in PDAC, and closely correlate with liver metastasis. The KGF/KGFR pathway may be a critical therapeutic target for PDAC metastasis.