Objectives: Emerging evidence has revealed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is implicated in the pathological processes of various kidney diseases, while its role in rhabdomyolysis-induced acute kidney injury (RIAKI) remains unknown. Methods: In this study, we applied renal tubular epithelial cell (RTEC)-specific NOX4 knockout (NOX4tecKO) and the NOX4 inhibitor GKT137831 to treat RIAKI in vivo and in vitro. In vivo, RIAKI mice was induced via intramuscular injection of 50% glycerol in hind thighs (8 ml/kg). In vitro, TCMK-1 (mouse kidney tubular epithelium cell line) cells were stimulated by 200 μM myoglobin. Serum and supernatant biochemical, inflammatory, and apoptotic parameters were measured and compared among groups. The activation of reactive oxygen species (ROS) and endoplasmic reticulum stress (ERS) signaling was also evaluated. Results: RTEC-specific NOX4 deficiency and GKT137831 treatment both protected against glycerin-induced renal function and pathology in RIAKI mice. Moreover, NOX4 inhibition reduced inflammation, decreased apoptosis of RTECs, and downregulated cleaved-Caspase3 expression. Surprisingly, similar results were obtained with GKT137831 treatment in vitro. Mechanistically, inhibition of NOX4 suppressed the generation of ROS and the expression of ERS-associated proteins at both RNA and protein levels, such as CHOP, GRP78, p‐JNK, and Caspase12 proteins. Conclusions: Collectively, genetic and pharmacological inhibition of NOX4 protects against RIAKI by mitigating ROS generation and suppressing activation of ERS signaling pathway, which attenuates inflammation and apoptosis. Targeting NOX4 may be a novel therapy for RIAKI.