Shear stress is a pathophysiologically relevant mechanical signal in cartilage biology and tissue engineering. Cyclooxygenase-2 (COX-2) is a pivotal proinflammatory enzyme, which is induced by mechanical loading-derived shear stress in chondrocytes. In this study, we investigated the transcriptional machinery and signaling pathway regulating shear-induced COX-2 expression in human chondrocytic cells. Deletion and mutation analysis of the human cox-2 promoter reveal that the CCAAT/enhancer-binding protein (C/EBP) and AP-1 predominantly contribute to the shear-induced cox-2 promoter activity. Supershift assays disclose that C/EBP, but not C/EBP or C/EBP, binds to the C/EBP site, whereas c-jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBP expression by c-jun, and the critical roles of both c-jun and C/EBP in shear-induced COX-2 synthesis. Our studies also indicate that Rac and to a lesser extent Cdc42 transactivate MEKK-1, which is in turn responsible for activation of mitogen-activated protein kinase kinase 7 (MKK7). MKK7 regulates c-Jun N-terminal kinase 2 (JNK2) activation, which in turn triggers the phosphorylation of c-jun that controls shear-mediated COX-2 upregulation in chondrocytes. Reconstructing the signaling network regulating shear-induced COX-2 expression and inflammation may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic interventions for arthritic disorders.