Tissue cells sense and respond to differences in substrate stiffness. In chondrocytes, it has been shown that substrate stiffness regulates cell spreading, proliferation, chondrogenic gene expression, and TGF-β signaling. But how the substrate stiffness together with soluble factors influences the mechanical properties of chondrocyte is still unclear. In this study, we cultured goat articular chondrocytes on polyacrylamide gels of 1, 11, and 90 kPa (Young’s modulus), and measured cellular stiffness, traction force, and response to stretch in the presence of TGF-β1 or IL-1β. We found that TGF-β1 increased cellular stiffness and traction force and enhanced the response to stretch, while IL-1β increased cellular stiffness, but lowered traction force and weakened the response to stretch. Importantly, the effects of TGF-β1 on chondrocyte mechanics were potent in cells cultured on 90 kPa substrates, while the effects of IL-1β were potent on 1 kPa substrates. We also demonstrated that such changes of chondrocyte mechanoresponse were due to not only the changes of actin cytoskeleton and focal adhesion, but also the alteration of chondrocyte extracellular matrix synthesis. Taken together, these results provide insights into how chondrocytes integrate physical and biochemical cues to regulate their biomechanical behavior, and thus have implications for the design of optimized mechanical and biochemical microenvironments for engineered cartilage.