BACKGROUND: Acute kidney injury (AKI) is a syndrome characterized by a precipitous decline in kidney function, posing a significant threat to patient survival. The role of RNA binding protein (RBP) in AKI remains insufficiently understood and we found an important RBP, Lgals3, that may mediate the progress of AKI. METHODS: Lgals3-/- mice, Nr4a1-/- mice and cross-linking immunoprecipitation and high-throughput sequencing were performed to examine the role of Lgals3 in AKI and the targeted binding proteins. RESULTS: Lgals3 expression was notably elevated in vivo and in vitro AKI models. Inhibition of Lgals3 mitigated kidney injury in both in vivo and in vitro AKI models. Conversely, kidney-specific overexpression of Lgals3 exacerbated kidney damage. Mechanistically, Lgals3 bound to the 3'-untranslated region of Nr4a1 via AAUAAA, resulting in upregulation of Nr4a1 and subsequent enhancement of Bap1 transcription, facilitating ferroptosis in AKI. Moreover, knockout of Nr4a1 or inhibition of the region of AAUAAA by antisense oligonucleotide (ASO) conferred protection against Lgals3-induced ferroptosis in AKI models. CONCLUSIONS: Lgals3 contributed to kidney injury by binding to the 3'UTR region of Nr4a1 via AAUAAA, leading to the activation of ferroptosis.