Natural extracellular matrix (ECM) mostly has a fibrous structure that supports and mechanically interacts with local residing cells to guide their behaviors. The effect of ECM elasticity on cell behaviors has been extensively investigated, while less attention has been paid to the effect of matrix fiber‐network plasticity at microscale, although plastic remodeling of fibrous matrix is a common phenomenon in fibrosis. Here, a significant decrease is found in plasticity of native fibrotic tissues, which is associated with an increase in matrix crosslinking. To explore the role of plasticity in fibrosis development, a set of 3D collagen nanofibrous matrix with constant modulus but tunable plasticity is constructed by adjusting the crosslinking degree. Using plasticity‐controlled 3D culture models, it is demonstrated that the decrease of matrix plasticity promotes fibroblast activation and spreading. Further, a coarse‐grained molecular dynamic model is developed to simulate the cell–matrix interaction at microscale. Combining with molecular experiments, it is revealed that the enhanced fibroblast activation is mediated through cytoskeletal tension and nuclear translocation of Yes‐associated protein. Taken together, the results clarify the effects of crosslinking‐induced plasticity changes of nanofibrous matrix on the development of fibrotic diseases and highlight plasticity as an important mechanical cue in understanding cell–matrix interactions.