Signaling pathways involved in oxidative stress-induced inhibition of osteoblast differentiation are not known. We showed in this report that H₂O₂ (0.1–0.2mM)-induced oxidative stress suppressed the osteoblastic differentiation process of primary rabbit bone marrow stromal cells (BMSC) and calvarial osteoblasts, manifested by a reduction of differentiation markers including alkaline phosphatase (ALP), type I collagen, colony-forming unit-osteoprogenitor (CFU-O) formation, and nuclear phosphorylation of Runx2. H₂O₂ treatment stimulated phospholipase C-γ1 (PLC-γ1), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB signaling but inhibited p38 mitogen-activated protein kinase (MAPK) activation. In the presence of 20μM PD98059 or 50μM caffeic acid phenethyl ester (CAPE), specific inhibitor for ERKs or NF-κB, respectively, could significantly reverse the decrease of above-mentioned osteoblastic differentiation markers elicited by H₂O₂ (0.1mM). Furthermore, PD98059 also suppressed H₂O₂-stimulated NF-κB signaling in this process. These data suggest that ERK and ERK-dependent NF-κB activation is required for oxidative stress-induced inhibition of osteoblastic differentiation in rabbit BMSC and calvarial osteoblasts.