Background: The seed of Cuscuta chinensis Lam. (Cuscuta, Convolvulaceae), is widely used in traditional Chinese medicine, with a variety of biological activity. However, the efficacy of C. chinensis has not been investigated in renal injury caused by sepsis. Objective: Our research was carried out to evaluate the effect of ethanol extract of C. chinensis seeds (ECCS) on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in Kunming mice, and explore its underlying mechanisms. Materials and Methods: ECCS was administered orally (10, 30, and 100 mg/kg bodyweight, once daily) to the AKI mice for 14 consecutive days. On the 14th day, LPS (7 mg/kg) was administered to induce AKI. Blood urea nitrogen (BUN), creatinine (Cr), cytokines, and antioxidant index were estimated. The protein phosphorylation was tested by Western blot. Results: Results indicated that ECCS (100 mg/kg) decreased LPS-induced augmented BUN by 58.54% (P < 0.001) and reduced Cr by 61.57% (P < 0.01), respectively. In addition, the evident reversion of renal pathological damage was observed in ECCS-pretreated mice. ECCS (100 mg/kg) also exhibited a reduction of cytokines (tumor necrosis factor-α, interleukin [IL]-1 β, and IL-6, P < 0.05 for all) in AKI mice. Furthermore, ECCS blocked the activation of the nuclear factor-κB (NF-κB) (p65 and inhibitor kappa B subunit) and reduced the toll-like receptors 4 (TLR4) expression. Conclusions: The protective effect of ECCS against LPS-induced AKI was at least partially associated with suppressing of a TLR4-NF-κB signaling pathway, which provides evidence of the renal protective function of C. chinensis extract.