Pancreatic cancer is characterized by very low survival rates because of high intrinsic resistance to conventional therapies. Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as one mechanism responsible for the limited benefit of radiotherapy in pancreatic cancer. In this study, we establish the role of heparanase-the only known mammalian endoglycosidase that cleaves heparan sulfate-in modulating the response of pancreatic cancer to radiotherapy. We found that clinically relevant doses of IR augment the invasive capability of pancreatic carcinoma cells in vitro and in vivo by upregulating heparanase. Changes in the levels of the transcription factor Egr-1 occurred in pancreatic cancer cells following radiation, underlying the stimulatory effect of IR on heparanase expression. Importantly, the specific heparanase inhibitor SST0001 abolished IR-enhanced invasiveness of pancreatic carcinoma cells in vitro, whereas combined treatment with SST0001 and IR, but not IR alone, attenuated the spread of orthotopic pancreatic tumors in vivo. Taken together, our results suggest that combining radiotherapy with heparanase inhibition is an effective strategy to prevent tumor resistance and dissemination, observed in many IR-treated pancreatic cancer patients. Further, the molecular mechanism underlying heparanase upregulation in pancreatic cancer that we identified in response to IR may help identify patients in which radiotherapeutic intervention may confer increased risk of metastatic spread, where antiheparanase therapy may be particularly beneficial.