Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1β stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1β signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE-/- mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.