[Abstract]? Objective: This study aims to elucidate whether ENO1 exacerbates renal injury by regulating the NF-κB signaling pathway to mediate M1 macrophage polarization. Methods:?An AKI model was established in C57BL/6 mice (n=12) via ischemia-reperfusion injury (IRI), with animals randomly assigned to Sham and IRI groups. Renal function was detected on the 3rd day after surgery. (serum creatinine, SCr; blood urea nitrogen, BUN) and observe the pathological changes of renal tissue.(HE and PAS staining). And the expressions of ENO1, kidney injury molecule-1 (KIM-1), and key proteins of the NF-κB pathway (p-p65, IκBα) were detected by IHC or Western blot, while the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) were quantified by ELISA. In vitro, human renal tubular epithelial cells (HK2) were exposed to tert-butyl hydroperoxide (t-BHP) to induce oxidative stress, followed by ENO1 knockdown or overexpression to assess HK2 cells impact on macrophage polarization markers (iNOS, CD206). Results: ENO1 expression was markedly upregulated in AKI mice, accompanied by deteriorated renal function（SCr、BUN）, elevated inflammatory cytokines, and aggravated renal tissue injury. Mechanistically, ENO1 activated the NF-κB pathway, evidenced by increased p-p65 levels and IκBα degradation, and promoted macrophage polarization toward the M1 phenotype (upregulated iNOS). Knockdown of ENO1 suppressed NF-κB activation, attenuated M1 polarization, and ameliorated inflammatory injury. In contrast, ENO1 overexpression further enhanced NF-κB activity and exacerbated inflammatory responses ( P < 0.05). Conclusion: IRI or oxidative stress can induce the upregulation of ENO1 expression in renal tubular cells. As a novel paracrine regulatory mediator, ENO1 promotes macrophage M1 polarization via activating the NF-κB pathway (with no significant effect on the M2 phenotype), thereby aggravating the inflammatory cascade in AKI. This suggests that ENO1 may serve as a potential therapeutic target for AKI, providing a novel insight into intervening in the inflammatory response.