Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a deadly illness characterized by persistent lung inflammation. This inflammation is commonly triggered by the bacterial endotoxin lipopolysaccharide (LPS). LPS activates the synthesis and release of inflammatory cytokines by binding TLR4 (toll-like receptor 4), which activates NF-κB (nuclear factor-κB). To understand the exact signaling mechanisms, researchers focused on two potentially involved proteins, ezrin and ROCK (Rho-associated coiled-coil containing protein kinase). Ezrin is a cross-linking protein that has been previously implicated in the activation of TLR4 signaling during LPS challenge, and ROCK is a kinase that may regulate the activity of ezrin-related proteins via phosphorylation. In cultured pulmonary alveolar epithelial cells, LPS induced ezrin phosphorylation, but this could be inhibited by blocking RhoA/ROCK. In co-immunoprecipitation assays, ezrin was associated with the signaling protein Syk constitutively and only associated with another signaling protein, MyD88 (myeloid differentiation factor 88) during LPS challenge. Downstream, the LPS-induced activation of p38 and nuclear NF-κB were dependent on ezrin and reducing ezrin expression or blocking ROCK activation reduced the LPS-related production of TNF-α, IL-1β, and HMGB1. While in vivo studies are needed, these results reveal a novel regulatory mechanism for LPS-induced production of pro-inflammatory cytokines.