The behavior of chondrocytes is regulated by multiple mechanical microenvironmental cues. During development and degenerative disease of articular cartilage, as an external signal, the extracellular matrix stiffness of chondrocytes changes significantly, but whether and how this biophysical cue affects biomechanical properties of chondrocytes remain elusive. In the present study, we designed supporting-biomaterials as mimics of native pericellular matrix to study the effect of matrix stiffness on chondrocyte morphology and F-actin distribution. Furthermore, the active mechanical behavior of chondrocytes during sensing and responding to different matrix stiffness was quantitatively investigated using atom force microscope technique and theoretical model. Our results indicated that stiffer matrix tends to increase the cell spreading area, the percentage of irregular cell shape distribution and mechanical parameters including elastic modulus (Eelastic), instantaneous modulus (E0), relaxed modulus (ER) and apparent viscosity (μ) of chondrocytes. Knowledge of matrix stiffness-dependent biomechanical behaviors of chondrocytes has important implications for optimizing matrix material and advancing chondrocyte-based applications for functional tissue engineering.