Lipopolysaccharide (LPS) or endotoxin can induce Toll-like receptor 4 signaling and cause microcirculatory dysfunction, which can lead to multiple organ dysfunction. The goal of this study was to investigate whether Toll-like receptor 4 antagonist, eritoran tetrasodium, can attenuate microcirculatory dysfunction in endotoxemic rats. Seventy-two male Wistar rats were divided into three groups as follows: control, LPS, and eritoran + LPS. These rats received laparotomy to exteriorize a segment of terminal ileum for microcirculation examination on intestinal mucosa, muscle, and Peyer patch. The rats in the eritoran + LPS group received 10 mg kg⁻¹ eritoran intravenously. The rats in the LPS and eritoran + LPS groups received 15 mg kg⁻¹ LPS intravenously. Microcirculatory blood flow intensity was measured by full-field laser perfusion imager. Total and perfused small-vessel densities, microvascular flow index, and heterogeneity index were investigated by sidestream dark-field video microscope. Our results revealed that eritoran restored the mean arterial pressure. At 240 min, the microcirculatory blood flow intensity was higher in the eritoran + LPS group than in the LPS group as follows: mucosa (1,094 [SD, 398] vs. 543 [SD, 163] perfusion unit [PU]; P < 0.001), muscle (752 [SD, 124] vs. 357 [SD, 208] PU; P < 0.001), and Peyer patch (961 [SD, 162] vs. 480 [SD, 201] PU; P < 0.001). Eritoran also attenuated endotoxin-induced elevation in the serum level of D-dimer. In conclusion, we have established a promising rat protocol to investigate the intestinal microcirculation in endotoxemia. Our data indicate that eritoran can reduce microcirculatory dysfunction in endotoxemic rats.