ABSTRACT Introduction: Chondrocytes perceive and respond to mechanical loading as signals that regulate their metabolism. Joint loading exposes chondrocytes to multiple modes of mechanical stress, including hydrostatic pressure; however, the mechanisms by which chondrocytes sense physiologically relevant levels of hydrostatic pressure are not well understood. We hypothesized that hydrostatic pressure is transduced to an intracellular signal through mechanosensitive membrane ion channels of chondrocytes. The goals of this study were to examine the effect of hydrostatic loading on the development of engineered cartilage tissue and the contribution of mechanosensitive ion channels on these hydrostatic loading effects. Methods: Using a 3D model of porcine chondrocytes in agarose, we applied specific chemical inhibitors to determine the role of transient receptor potential (TRP) ion channels TRPV1, TRPV4, TRPC3, and TRPC1 in transducing hydrostatic pressure. Results: Hydrostatic loading caused a frequency and magnitude-dependent decrease in sulfated glycosaminoglycans (S-GAG), without changes in DNA content. Inhibiting TRPC3 and TRPV4 decreased S-GAG content; however, only the inhibition of TRPV1 partially attenuated the hydrostatic loading-induced reduction in S-GAG content. Conclusions: Our findings indicate that TRPV1 may serve as a transducer of hydrostatic pressure in chondrocytes, and provide further support for the role of TRPV4 in regulating chondrocyte anabolism, as well as initial evidence implicating TRPC3 in chondrogenesis. These findings add to our further understanding of the chondrocyte “channelome” and suggest that a range of ion channels mediate the transduction of different biophysical stimuli such as hydrostatic pressure, membrane stretch, or osmotic stress.