Inappropriate mechanical ventilation (MV) can result in ventilator-induced lung injury (VILI). Probing mechanisms of VILI and searching for effective methods are current areas of research focus on VILI. The present study aimed to probe into mechanisms of endothelial microparticles (EMPs) in VILI and the protective effects of Tetramethylpyrazine (TMP) against VILI. In this study, C57BL/6 and TLR4KO mouse MV models were used to explore the function of EMPs associated with p21 activated kinases-4 (PAK-4) in VILI. Both the C57BL/6 and TLR4 KO groups were subdivided into a mechanical ventilation (MV) group, a TMP + MV group, and a control group. After four hours of high tidal volume (20 ml/kg) MV, the degree of lung injury and the protective effects of TMP were assessed. VILI inhibited the cytoskeleton-regulating protein of PAK4 and was accompanied by an increased circulating EMP level. The intercellular junction protein of β-catenin was also decreased accompanied by a thickening alveolar wall, increased lung W/D values, and neutrophil infiltration. TMP alleviated VILI via decreasing circulating EMPs, stabilizing intercellular junctions, and alleviating neutrophil infiltration.