Chondrocytes change their morphology and dedifferentiate to fibroblast-like cell during in vitro expansion culture. The phenotype variation may have some effect on the mechanical properties of living chondrocytes. This study was performed to determine the effect of the living chondrocytes’ passage number on their stiffness using atomic force microscopy (AFM). We used freshly isolated (P0) and serially passaged (P1-P5) bovine articular chondrocytes (BACs) and compared the results of Young's modulus measurements with changes in the morphology of the cells. We found that the BACs phenotype variation was accompanied with a decrease in the elastic modulus of the living cells. The change in stiffness was most rapid for the first two passages (P0–P2). The comparison of the mechanical properties of chondrocytes with intact cellular membranes and cells with removed membranes enabled us to conclude that the cellular membrane can play a significant role in the mechanical properties of chondrocytes.