TNF-α, one of the most potent pro-inflammatory cytokines, plays a critical role in inhibition of osteoblast differentiation and bone regeneration in persistent inflammatory microenvironment. To explore the mechanism, quantitative proteomics based on iTRAQ and MRM was employed. The results showed 6 proteins involved in BMP-2 induced osteoblast differentiation inhibition by TNF-α: Periostin, Protein S100-A4, ATPase inhibitor, Cytochrome b5, SERCA3, and ELP2. The altered proteins were involved in molecular transport, tissue development, energy metabolism, and inflammation. One specific protein, ELP2 (STAT3-interacting protein 1, StIP1) up-regulated in the inhibition of osteoblast differentiation by TNF-α was verified to play a critical role in STAT3 pathway. Overexpression or knockdown of ELP2 in C2C12 and MC3T3-E1 cells affected osteoblast differentiation inhibition induced by TNF-α. These results highlight the function of ELP2 in inflammatory microenvironment, ELP2 up-regulation and STAT3 pathway activation may down-regulate BMPR2, then BMP-2 was blocked and osteoblast differentiation inhibited. The protein-expression profile revealed here should offer at least partly new clues to understand the mechanism of osteoblast differentiation inhibition by inflammation. Persistent inflammation is always associated with osteogenesis and affects this balance to reduce bone mass including traumatic open bone fracture, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but the cellular mechanisms are not fully elucidated. Tumor necrosis factor-α (TNF-α) is one of the most potent pro-inflammatory cytokines and is known to be a catabolic factor in these inflammatory reaction of diseases. We show for the first time using proteomics methods that in inflammatory microenvironment, osteoblast differentiation will be inhibited by TNF-α induced ELP2 up-regulation and STAT3 pathway activation. Our results are significant since they point to targeting ELP2 activity as a novel therapeutic option to limit the inhibition of osteoblast differentiation by inflammatory microenvironment. Copyright © 2014 Elsevier B.V. All rights reserved.