Objective: This study was designed to observe the effect of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-kappa B) pathway activity on sepsis-associated acute kidney injury (SA-AKI), thereby providing new considerations for the prevention and treatment of SA-AKI. Methods: The rats were divided into Sham, cecal ligation and puncture (CLP), CLP + vehicle, and CLP + TAK-242 groups. Except the Sham group, a model of CLP-induced sepsis was established in other groups. After 24 h, the indicators related to kidney injury in blood samples were detected. The pathological changes in the kidneys were observed by hematoxylin-eosin staining, and tubular damage was scored. Oxidative stress-related factors, mitochondrial dysfunction-related indicators in each group were measured; the levels of inflammatory factors in serum and kidney tissue of rats were examined. Finally, the expression of proteins related to the TLR4/NF-kappa B signaling pathway was observed by western blot. Results: Compared with the CLP + vehicle and CLP + TAK-242 groups, the CLP + TAK-242 group reduced blood urea nitrogen (BUN), creatinine (Cr), cystatin-C (Cys-C), reactive oxygen species (ROS), malondialdehyde (MDA), and inflammatory factors levels (p < 0.01), as well as increased superoxide dismutase (SOD) activity of CLP rats (p < 0.01). Additionally, TAK-242 treatment improved the condition of CLP rats that had glomerular and tubular injuries and mitochondrial disorders (p < 0.01). Further mechanism research revealed that TAK-242 can inhibit the TLR4/NF-kappa B signaling pathway activated by CLP (p < 0.01). Above indicators after TAK-242 treatment were close to those of the Sham group. Conclusion: TAK-242 can improve oxidative stress, mitochondrial dysfunction, and inflammatory response by inhibiting the activity of TLR4/NF-kappa B signaling pathway, thereby preventing rats from SA-AKI.