PEGylated human hyaluronidase (PEGPH20) has been developed to enzymatically deplete tumor hyaluronan. The purpose of this study is to investigate physiologic and metabolic changes in pancreatic adenocarcinoma xenograft after PEGPH20 treatment by using multi-modal imaging and further determine the utility of PEGPH20 as radiosensitizer. PEGPH20 significantly increased intratumor pO2 and blood volume and decreased glycolytic flux assessed by EPRI, USPIO-MRI, and hyperpolarized 13C-MRI, respectively. PEGPH20 also enhanced treatment effect of radiotherapy in vivo. The results validated the utility of the imaging methods to non-invasively monitor changes in the tumor microenvironment and predicted the radiosensitizing effect upon hyaluronan depletion.