Respiratory syncytial virus (RSV) is the major cause of bronchiolitis in infants and a common feature of RSV infections is increased lung permeability. The accumulation of fluid in the infected lungs is caused by changes in the endothelial and epithelial membrane integrity. However, the exact mechanisms of viral-induced fluid extravasation remain unclear. Here, we report that infection of human epithelial cells with RSV results in significant epithelial membrane barrier disruption as assessed by a decrease in trans-epithelial resistance (TEpR). This decrease in TEpR, which indicate changes in paracellular permeability, was mediated by marked cellular cytoskeletal rearrangement. Importantly, the decrease in TEpR was attenuated by using p38 MAPK inhibitors (SB203580), but was partially affected by c-Jun N-terminal kinase (JNK) inhibitor SP600125. Interestingly, treatment of A549 cells with MEK1/2 inhibitor (UO126) led to a decrease in TEpR in the absence of RSV infection. The changes in TEpR were concomitant with an increase in Hsp27 phosphorylation and with actin microfilament rearrangement. Thus, our data suggest that p38 MAPK and Hsp27 are required for RSV induction of human epithelial membrane permeability.