Aims: Nuclear factor (NF)-kappa B signaling pathway contributes to the pathogenesis of lung fibrosis. However, the expression and roles of NF-kappa B p65 subunit in fibroblasts under fibrotic conditions have not been studied. We checked the expression of p65 in lung tissue and fibroblasts from bleomycin (BLM) challenged mice and investigated the roles of p65 in human lung fibroblast differentiation during transforming growth factor beta (TGF-beta) challenge. Main methods: A murine model of BLM-induced lung fibrosis and in vitro cultures of lung fibroblast from human and mouse were used to study the expression and role of p65 during fibrogenesis. The si-RNA knockdown strategy was used to check the mechanism of TGF-beta induced p65 expression and roles of p65 during fibroblast differentiation. Key findings: We found that the expression of p65 was significantly increased in lung tissue, fibrotic foci and fibroblast from BLM-challenged mice. In vitro, TGF-beta stimulated p65 expression, nuclear translocation and cell differentiation in human lung fibroblast; knockdown smad3 expression inhibited TGF-beta-induced p65 expression and differentiation in human lung fibroblast Additionally, knockdown the expression of p65 suppressed TGF-beta-induced differentiation and p65 translocation to nuclei in human lung fibroblast. Significance: These data suggested that p65 expression is up-regulated in lung fibroblast during differentiation through TGF-beta/smad3 pathway and synergistically regulates TGF-beta induced differentiation of lung fibroblasts. (C) 2014 Elsevier Inc. All rights reserved.