The expression of cyclooxygenase-2 (COX-2) is a characteristic response to inflammation and can be inhibited with sodium salicylate. TNF-α plus IFN-γ can induce extracellular signal-regulated kinase (ERK), IKK, IκB degradation and NF-κB activation. The inhibition of the ERK pathway with selective inhibitor, PD098059, blocked cytokine-induced COX-2 expression and PGE2 release. Salicylate treatment inhibited COX-2 expression induced by TNF-α/IFN-γ and regulated the activation of ERK, IKK and IκB degradation and subsequent NF-κB activation in MC3T3E1 osteoblasts. Furthermore, antioxidants such as catalase, N-acetyl-cysteine or reduced glutathione attenuated COX-2 expression in combined cytokines-treated cells, and also inhibited the activation of ERK, IKK and NF-κB in MC3T3E1 osteoblasts. In addition, TNF-α/IFN-γ stimulated ROS release in the osteoblasts. However, salicylate had no obvious effect on ROS release in DCFDA assay. The results showed that salicylate inhibited the activation of ERK and IKK, IκB degradation and NF-κB activation independent of ROS release and suggested that salicylate exerts its anti-inflammatory action in part through inhibition of ERK, IKK, IκB, NF-κB and resultant COX-2 expression pathway.