The success of replicating adenoviruses for cancer therapy is limited by inefficient virus delivery and poor distribution within the tumor mass. Stromal matrix within the tumor may hinder the free cell-to-cell spread of the virus. In this study, in vitro cell culture experiments showed that collagen I blocked the passage of an adenoviral vector through a membrane. On the basis of reports of the effective collagen I-degrading activity of matrix metalloproteinase-8 (MMP-8), we constructed an adenovirus to express the MMP-8 transgene (AdMMP8). A549 cells infected in vitro with AdMMP8 did not show altered growth but were able to modify a fibrillar collagen substrate to allow viral diffusion. Further, AdMMP8 did not affect replication of the wild-type virus (Adwt300). Established human A549 lung cancer and BxPC-3 pancreatic cancer xenograft tumors that were injected with Adwt300 together with the non-replicating AdMMP8 virus showed significantly reduced growth compared with control tumors. Histochemical analysis showed reduced amounts of collagen within necrotic areas of MMP-8-injected tumors compared with controls. These results demonstrate that intra-tumoral expression of MMP-8 is a possible strategy for improving viral spread and improving the oncolytic activity of replicating adenovirus.