Recently, we demonstrated that intratracheal transplantation of human umbilical cord blood- derived mesenchymal stem cells (MSCs) attenuatesEscherichia (E) coli- induced acute lung injury primarily by down- modulating inflammation and enhancing bacterial clearance iQn mice. This study was performed to elucidate the mechanism underlying the antibacterial effects of MSCs. The growth ofE. coli in vitrowas significantly inhibited only by MSCs or their conditioned medium with bacterial preconditioning, but not by fibroblasts or their conditioned medium. Microarray analysis identified significant up- regulation of toll- like receptors (TLR)- 2 and TLR- 4, and β- defensin 2 (BD2) in MSCs compared with fibroblasts afterE. coliexposure. The increased BD2 level and thein vitroantibacterial effects of MSCs were abolished by specific antagonist or by siRNA- mediated knockdown of TLR- 4, but not TLR- 2, and restored by BD2 supplementation. Thein vivodown- modulation of the inflammatory response and enhanced bacterial clearance, increased BD2 secretion and the resultant protection againstE. coli- induced pneumonia observed only with MSCs, but not fibroblasts, transplantation in mice, were abolished by knockdown of TLR- 4 with siRNA transfection. Our data indicate that BD2 secreted by the MSCs via the TLR- 4 signalling pathway is one of the critical paracrine factors mediating their microbicidal effects againstE. coli, bothin vitroandin vivo. Furthermore, TLR- 4 from the transplanted MSCs plays a seminal role in attenuatingin vivo E. coli- induced pneumonia and the ensuing acute lung injury through both its anti- inflammatory and antibacterial effects.