Dynamic mechanical loading can enhance the formation of engineered cartilage, potentially through secondary biophysical effects such as changes in interstitial osmolarity. This study examined the effects of daily osmotic loading, as well as direct activation of the osmosensitive ion channel TRPV4, on the biochemical and functional properties of chondrocyte-laden cartilage constructs. Osmotic loading, as well as exposure to the TRPV4-specific agonist GSK1016790A, enhanced extracellular matrix (ECM) accumulation, and TRPV4 activation enhanced the functional properties of the constructs. This study implicates the Ca++-permeable TRPV4 ion channel in the metabolic response of articular chondrocytes to osmotic and mechanical loading. Furthermore, these results suggest that targeting TRPV4, either directly with channel agonists, or indirectly via osmotic loading, may provide a novel strategy for enhancing tissue engineered cartilage construct maturation.