Chondrocytes regulate the composition of cartilage extracellular matrix in response to mechanical signals, but the intracellular pathways involved in mechanotransduction are still being defined. Mitogen-activated protein kinase (MAPK) pathways are activated by static and dynamic compression of cartilage, which simultaneously induce intratissue fluid flow, pressure gradients, cell, and matrix deformation. First, to determine whether cell and matrix deformation alone could induce MAPK activation, we applied dynamic shear to bovine cartilage explants. Using Western blotting, we measured ERK1/2 and p38 activation at multiple time points over 24 h. Distinct activation time courses were observed for different MAPKs: a sustained 50% increase for ERK1/2 and a delayed increase in p38 of 180%. We then investigated the role of MAPK activation in mechano-induced chondrocyte gene expression. Cartilage explants were preincubated with inhibitors of ERK1/2 and p38 activation before application of 1-24 h of three distinct mechanical stimuli relevant to in vivo loading (50% static compression, 3% dynamic compression at 0.1 Hz, or 3% dynamic shear at 0.1 Hz). mRNA levels of selected genes involved in matrix homeostasis were measured using real-time PCR and analyzed by k-means clustering to characterize the time- and load-dependent effects of the inhibitors. Most genes examined required ERK1/2 and p38 activation to be regulated by these loading regimens, including matrix proteins aggrecan and type II collagen, matrix metalloproteinases MMP13, and ADAMTS5, and transcription factors downstream of the MAPK pathway, c-Fos, and c-Jun. Thus, we demonstrated that the MAPK pathway is a central conduit for transducing mechanical forces into biological responses in cartilage.