Extracellular matrix (ECM) is a dynamic and complex environment regulating the cell fate and behavior. It is characterized by biophysical and biochemical properties specific for each tissue. Interestingly, hydrogels can serve as exceptional artificial cellular microenvironments as they can be designed to mimic the key features of the native ECM. They are valuable tools to understand how cells respond to complex microenvironments in normal and pathologic conditions. However, unlike the highly dynamic structure of ECM, nearly all of the conventional hydrogel platforms are primarily static and lack the dynamic properties of native extracellular matrices. Thus, it is necessary to develop dynamic hydrogels to better understand the mechanisms by which dynamic changes of ECM contribute to biological processes. Stiffness is one of the significant dynamic components of ECM which must be appropriately mimicked over time . In this review, we cover recent advances in engineering strategies to make cell laden hydrogels with temporally tunable stiffness. We also highlight the applications of these hydrogel systems in biomedicine focusing on specific examples in cancer, cardiovascular system, tissue fibrosis and stem cell research. Finally, the challenges regarding the development and application of cell laden hydrogels with temporally tunable stiffness are proposed.