〔Abstract〕 Objective To investigate the effect of receptor of activated protein kinase C 1 (RACK1) on lipopolysaccharide (LPS)-induced barrier function in rat pulmonary microvascular endothelial cells (RPMVEC) and its interplay with the Sonic hedgehog (SHH) signaling pathway. Methods RPMVEC were cultured in vitroand randomly divided into si-NC, si-NC+LPS, si-RACK1, si-RACK1+LPS, si-RACK1+LPS+Vismodegib and Vismodegib+SAG groups. The RACK1 of RPMVEC was silenced by small interfering RNA (siRNA) technology and the cells were treated with LPS(10 mg/L), SHH signaling pathway inhibitor (Vismodegib) (20 μmol/L) and SHH signaling pathway agonist (SAG) (1 μmol/L). Following the intervention, the expressions of RACK1 and caveolin-1 in RPMVEC were detected by immunofluorescence, while the transendothelial electrical resistance (TEER) was evaluated using the method of Transwell, and the expression levels of RACK1, glioma-associated oncogene homolog 1 (Gli-1) and caveolin-1 were detected by Western blot. Results Silencing RACK1 significantly increased the TEER value of RPMVEC induced by LPS ( P<0.05). The expression of caveolin-1 decreased ( P<0.05), and the expression of Gli-1 increased ( P<0.05). Inhibiting the SHH signaling pathway could reverse the increased TEER value of LPS-induced RPMVEC caused by silencing RACK1 ( P<0.05), and the expressions of RACK1 and caveolin-1 increased ( P<0.05). Activation of the SHH signaling pathway increased the TEER value of LPS-induced RPMVEC caused by silencing RACK1 (P<0.05), and the expressions of RACK1 and caveolin-1 decreased ( P<0.05). Conclusion RACK1 plays a role in LPS-induced hyperpermeability of RPMVEC, and its effect may be achieved by modulating the SHH signaling pathway and caveolin-1.