Articular cartilage, composed of collagen type II as a major extracellular matrix and chondrocyte as a unique cell type, is a specialized connective tissue without blood vessels, lymphatic vessels and nerves. This distinctive characteristic of articular cartilage determines its very limited ability to repair when damaged. It is well known that physical microenvironmental signals regulate many cell behaviors such as cell morphology, adhesion, proliferation and cell communication even determine chondrocyte fate. Interestingly, with increasing age or progression of joint diseases such as osteoarthritis (OA), the major collagen fibrils in the extracellular matrix of articular cartilage become larger in diameter, leading to stiffening of articular tissue and reducing its resistance to external tension, which in turn aggravates joint damage or progression of joint diseases. Therefore, designing a physical microenvironment closer to the real tissue and thus obtaining data closer to the real cellular behaviour, and then revealing the biological mechanisms of chondrocytes in pathological states is of crucial importance for the treatment of OA disease. Here we fabricated micropillar substrates with the same topology but different stiffnesses to mimic the matrix stiffening that occurs in the transition from normal to diseased cartilage. It was first found that chondrocytes responded to stiffened micropillar substrates by showing a larger cell spreading area, a stronger enhancement of cytoskeleton rearrangement and more stability of focal adhesion plaques. The activation of Erk/MAPK signalling in chondrocytes was detected in response to the stiffened micropillar substrate. Interestingly, a larger nuclear spreading area of chondrocytes at the interface layer between the cells and top surfaces of micropillars was observed in response to the stiffened micropillar substrate. Finally, it was found that the stiffened micropillar substrate promoted chondrocyte hypertrophy. Taken together, these results revealed the cell responses of chondrocytes in terms of cell morphology, cytoskeleton, focal adhesion, nuclei and cell hypertrophy, and may be beneficial for understanding the cellular functional changes affected by the matrix stiffening that occurs during the transition from a normal state to a state of osteoarthritis.