Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3 epsilon protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3 epsilon. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3 epsilon-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3 epsilon signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3 epsilon. Direct interaction between CD13 and 14-3-3 epsilon was then demonstrated by surface plasmon resonance. Using labeled 14-3-3 epsilon, we also found that 14-3-3 epsilon binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3 epsilon might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3 epsilon-CD13 interaction could be a new therapeutic target in osteoarthritis.