As cell function and phenotype can be directed by the mechanical characteristics of the surrounding matrix, hydrogels have become important platforms for cell culture systems, with properties that can be tuned by external stimuli such as divalent cations, enzymatic treatment and pH. However, many of these stimuli can directly affect cell behaviour, making it difficult to distinguish purely mechanical signalling events. This study reports on the development of a hydrogel that incorporates photoswitchable crosslinkers, which can reversibly alter their stiffness upon irradiation with the appropriate wavelength of light. Furthermore, this study reports the response of bone marrow derived mesenchymal stem cells (MSCs) on these hydrogels that were stiffened systematically by irradiation with blue light. The substrates were shown to be non-cytotoxic, and crucially MSCs are not affected by blue light exposure. Time-resolved analysis of cell morphology showed characteristic cell spreading and increased aspect ratios in response to greater substrate stiffness. This hydrogel provides a platform to study mechano-signalling in cells responding to dynamic changes in stiffness, offering a new way to study mechanotransduction signalling pathways and biological processes with implicit changes to tissue mechanics, such as development, ageing and fibrosis.