Osteoarthritis (OA) is a whole-joint disease characterized by progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13/aminopeptidase N (CD13/APN) in the chondrocyte response to 14-3-3ε. After identifying CD13/APN in chondrocytes, we knocked down CD13/APN by siRNA and blocking antibodies in articular chondrocytes. 14-3-3ε–induced (MMP-3) and MMP-13 was significantly reduced with CD13/APN knockdown, which suggests its critical role in 14-3-3ε signal transduction. APN activity was identified in chondrocytes, but the activity was unchanged after stimulation of chondrocytes with 14-3-3ε. Direct interaction between CD13/APN and 14-3-3ε was then demonstrated by surface plasmon resonance (SPR). Using labeled 14-3-3ε, we showed also that 14-3-3ε binds to chondrocytes surface dependently of CD13/APN. All together, these results suggest that 14-3-3ε may directly bind to CD13/APN which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in OA. The 14-3-3ε–CD13/APN interaction could be a novel therapeutic target for exploration in OA.