<p>Diabetic nephropathy (DN) is a global health threat with limited therapeutic interventions. Irisin, a myokine derived from FNDC5, has been implicated in glucose homeostasis and anti-diabetic effects; however, its precise mechanistic role in glucose metabolism regulation remains elusive. This study elucidates the specific role of Irisin in mitigating diabetic renal tubular epithelial injury, with a focus on its regulatory mechanisms under glucotoxic conditions. Utilizing streptozotocin (STZ)-induced type 1 diabetes (T1D) mouse models and high glucose (HG)-stimulated HK-2 cells, we demonstrated that STZ-induced DN mice exhibited renal dysfunction, oxidative stress, and iron accumulation. Sustained HG exposure downregulated glutathione peroxidase 4 (GPX4) and xCT while upregulating Ptgs2 and FPN1, indicative of Ferroptosis initiation. Irisin treatment significantly attenuated these pathological changes and ameliorated renal tubular epithelial injury. Mechanistically, this protective effect was mediated through the activation of high-mobility group box-1 (HMGB1), a damage-associated regulator, as observed in both in <i>vivo</i> and in <i>vitro</i> studies. Furthermore, we identified the nuclear translocation of Nrf2 and its downstream target GPX4 in <i>vitro</i>. Specific interference with Nrf2, through both knockdown and overexpression experiments under HG conditions, further demonstrated Irisin's regulatory role on HMGB1. This was validated by assessing tubular epithelial cell viability, alongside cellular levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and Fe²⁺ content. Notably, Nrf2 co-transfection nullified the protective effects of Irisin, exacerbating Ferroptosis markers. Collectively, our findings reveal that Irisin protects against glucotoxicity-induced renal injury by inhibiting tubular epithelial Ferroptosis via the HMGB1/Nrf2/GPX4 axis, thereby proposing a novel therapeutic target for DN.</p>