Objective To determine if enolase 1 (ENO1) exacerbates renal injury by regulating the nuclear factor (NF)-κB signaling pathway to mediate M1 macrophage polarization. Methods An acute kidney injury (AKI) model was established in C57BL / 6 mice ( n= 12) via ischemia-reperfusion injury ( IRI). Mice were assigned randomly to Sham and IRI groups. Renal function was detected on the 3rd day after surgery by measuring serum creatinine ( SCr) and blood urea nitrogen ( BUN), and pathological changes in renal tissue were detected by hematoxylin-eosin and periodic acid-Schiff staining. Expression levels of ENO1, kidney injury molecule-1 (KIM-1),and key proteins in the NF-κB pathway ( phospho-p65, nuclear factor-κB inhibitor α ( IκBα)) were detected by immunohistochemistry or Western blot, and levels of inflammatory cytokines ( tumor necrosis factor ( TNF)-α,interleukin (IL)-1β, IL-6) were quantified by enzyme-linked immunosorbent assay. Human renal tubular epithelial cells ( HK2 ) were exposed to tert-butyl hydroperoxide in vitro to induce oxidative stress, followed by ENO1 knockdown or overexpression to assess the impact on macrophage polarization markers (inducible nitric oxide synthase (iNOS), CD206). Results ENO1 expression was markedly upregulated in AKI mice(P<0. 05), accompanied by deteriorated renal function ( SCr, BUN, P<0. 001), elevated inflammatory cytokines, and aggravated renal tissue injury(P<0. 001). Mechanistically, ENO1 activated the NF-κB pathway, evidenced by increased p-p65 levels and IκBα degradation ( P<0. 01, P<0. 001 ), and promoted macrophage polarization towards the M1 phenotype (upregulated iNOS). Knockdown of ENO1 suppressed NF-κB activation ( P<0. 05, P< 0. 001), attenuated M1 polarization, and ameliorated inflammatory injury(P<0. 01), while ENO1 overexpression further enhanced NF-κB activity and exacerbated inflammatory responses. Conclusions 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 for intervening in the inflammatory response.