Transitions in mechanical properties between different tissue scaffolds, such as extracellular matrices (ECMs), are common in the human body and influence cellular orientation. For example, in teeth, the abrupt transition between soft (approx5 kPa) dental pulp and stiff (more than100 kPa) dentin directs the alignment of odontoblasts. In this study, hydrogels, with defined mechanical transitions were used to evaluate the phenotypic response of fibroblasts to either a soft/soft (3 kPa/3 kPa) or soft/stiff (3 kPa/200 kPa) interface. Near the soft/soft interface fibroblasts aligned in a more parallel fashion to the interface (11 degree angle to the interface), compared to the more perpendicular fashion (69 degree angle to the interface) observed at the soft-stiff interface. Notably, regions of alignment also showed an increased proportion of alpha smooth muscle actin (alpha-SMA)-positive cells, indicating myofibroblast differentiation in response to the mechanical transition. These findings demonstrate that modifying a biomaterial by introducing an interface alters the bioactivity of responding cells solely through mechanical mechanisms, without any change to the cells biochemical environment.