Disturbed blood flow induces inflammatory gene expression in endothelial cells, which promotes atherosclerosis. Flow stimulates the proinflammatory transcription factor nuclear factor (NF)-κB through integrin- and Rac-dependent production of reactive oxygen species (ROS). Previous work demonstrated that NF-κB activation by flow is matrix-specific, occurring in cells on fibronectin but not collagen. Activation of p21-activated kinase (PAK) followed the same matrix-dependent pattern. We now show that inhibiting PAK in cells on fibronectin blocked NF-κB activation by both laminar and oscillatory flow in vitro and at sites of disturbed flow in vivo. Constitutively active PAK rescued flow-induced NF-κB activation in cells on collagen. Surprisingly, PAK was not required for flow-induced ROS production. Instead, PAK modulated the ability of ROS to activate the NF-κB pathway. These data demonstrate that PAK controls NF-κB activation by modulating the sensitivity of cells to ROS.