The aim of this study was to investigate the protective effects of Moringa oleifera leaf polysaccharide (MLP) on rhabdomyolysis-induced acute kidney injury (RM-AKI) and the potential mechanisms of such effects. A mouse RM-AKI model and a ferrous myoglobin-induced injury model were established in renal tubular epithelial cells (HK-2). Biochemical kits were used to measure the levels of creatinine (Cr), blood urea nitrogen (BUN), and creatine kinase (CK) in mouse serum, as well as cytokines (IL-1β, IL-6, and TNF-α) in kidney tissue. Furthermore, hematoxylin and eosin staining and TUNEL staining were used to evaluate pathological changes and apoptosis in kidney tissue. Western blotting was performed to evaluate the expression of key proteins in both kidney tissue and HK-2 cells. The results revealed that, compared with the model group, the levels of Cr, BUN, CK, IL-1β, IL-6, and TNF-α in the high-dose MLP-treated mice decreased by 67.87%, 43.03%, 21.39%, 31.05%, 54.20%, and 51.66%, respectively (P<0.001). In addition, HK-2 apoptosis was significantly inhibited, and oxidative stress levels notably decreased in ferrous myoglobin-induced HK-2 cells. A significant downregulation in the expression of TLR4, P-p65/p65, and cleaved caspase-3, both in vivo and in vitro, was observed after MLP treatment. These results indicate that MLP has a significant protective effect on RM-AKI, which provide a scientific basis for the clinical treatment of patients with RM-AKI.