Lipopolysaccharide (LPS)-induced microvascular hyperpermeability and hemorrhage play a key role in the development of sepsis, attenuation of which might be an important strategy to prevent sepsis. However, present clinical therapies are proved to be inefficiency in improving the final survival rate for the patients with sepsis. Catalpol, an iridoid glycoside extracted from the roots of Rehmannia, has been reported able to protect against LPS-induced acute lung injury through Tool-like Receptor-4 (TLR-4) mediated NF-B signaling pathway. However, it is still unknown whether Catalpol is effective to ameliorate the LPS-induced microvascular disorder. The present study was aimed to investigate the impact of Catalpol on LPS-induced mesenteric microvascular disorder and its underlying mechanism. Male Wistar rats were challenged by infusion of LPS (10 mg/kg/h) through left femoral vein for 120 min. Post-treatment with Catalpol (10 mg/kg) alleviated the LPS-induced microvascular hyperpermeability and hemorrhage, reduced mortality, ameliorated the alteration in distribution of claudin-5 and JAM-1, and the degradation of collagen IV and laminin, attenuated the increase of TLR-4 level, phosphorylations of Src tyrosine kinase, phosphatidyl inositol 3-kinase, focal adhesion kinase, and Cathepsin B activation. Inhibition of TLR-4 and Src each simulated some but not all effects Catalpol exerted. Besides, surface plasmon resonance showed that Catalpol could directly bind to TLR-4 and Src. These results demonstrated that Catalpol was able to ameliorate the LPS-induced microvascular barrier damage and hemorrhage by targeting both TLR-4 and Src thus attenuating the phosphorylation of Src kinase, phosphatidyl inositol 3-kinase and focal adhesion kinase, as well as Cathepsin B activation.