The progression of cancer is often accompanied by changes in the mechanical properties of an extracellular matrix. However, limited efforts have been made to reproduce these biological events in vitro. To this end, this study demonstrates that matrix remodeling caused by matrix metalloproteinase (MMP)-1 regulates phenotypic activities and modulates radiosensitivity of cancer cells exclusively in a 3D matrix. In this study, hepatocarcinoma cells are cultured in a collagen-based gel tailored to present an elastic modulus of approximate to 4.0 kPa. The subsequent exposure of the gel to MMP-1 decreases the elastic modulus from 4.0 to 0.5 kPa. In response to MMP-1, liver cancer cells undergo active proliferation, downregulation of E-cadherin, and the loss of detoxification capacity. The resulting spheroids are more sensitive to radiation than the spheroids cultured in the stiffer gel not exposed to MMP-1. Overall, this study serves to better understand and control the effects of MMP-induced matrix remodeling.