Acute kidney injury causes significant morbidity and mortality. This experimental animal study investigated the simultaneous impact of iron and vitamin D on acute kidney injury induced by iohexol, an iodinated, non-ionic monomeric radiocontrast agent in Wistar rats. Out of 36 healthy male Wistar rats, saline was injected into six control rats (group 1) and iohexol into the remaining 30 experimental rats (groups 2 to 6 comprising six rats each). Biochemical, renal histological changes, and gene expression of iron-regulating proteins and 1 α-hydroxylase were analyzed. Urinary neutrophil gelatinase-associated lipocalin (NGAL), serum creatinine, urine protein, serum and urine catalytic iron, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and tissue lipid peroxidation were assayed. Rats injected with iohexol showed elevated urinary NGAL (11.94 ± 6.79 ng/mL), serum creatinine (2.92 ± 0.91 mg/dL), and urinary protein levels (11.03 ± 9.68 mg/mg creatinine) together with histological evidence of tubular injury and iron accumulation. Gene expression of iron-regulating proteins and 1 α-hydroxylase was altered. Serum and urine catalytic iron levels were elevated (0.57 ± 0.17; 48.95 ± 29.13 µmol/L) compared to controls (0.49 ± 0.04; 20.7 ± 2.62 µmol/L, P ' 0.001). Urine catalytic iron positively correlated with tissue peroxidation (r = 0.469, CI 0.122 to 0.667, P = 0.004) and urinary NGAL (r = 0.788, CI 0.620 to 0.887, P ' 0.001). 25-hydroxyvitamin D3 (61.58 ± 9.60 ng/mL) and 1,25-dihydroxyvitamin D3 (50.44 ± 19.76 pg/mL) levels increased simultaneously. In a multivariate linear regression analysis, serum iron, urine catalytic iron, and tissue lipid peroxidation independently and positively predicted urinary NGAL, an acute kidney injury biomarker. This study highlights the nephrotoxic potential of catalytic iron besides demonstrating a concurrent induction of vitamin D endogenously for possible renoprotection in acute kidney injury. Impact statement: This work provides in-depth insights on catalytic iron-induced cytotoxicity and the resultant triggering of endogenous vitamin D synthesis in experimental acute kidney injury. Our results reveal significantly elevated levels of catalytic iron culminating in oxidant-mediated renal injury and a concomitant increase in 1,25-dihdyroxyvitamin D3 levels. Also, changes in other iron-related proteins including transferrin, ferritin, and hepcidin were observed both in the serum as well as in their mRNA expression. We consider all these findings vital since no connection between catalytic iron and vitamin D has been established so far. Furthermore, we believe that this work provides new and interesting results, with catalytic iron emerging as an important target in ameliorating renal cellular injury, possibly by timely administration of vitamin D. It also needs to be seen if these observations made in rats could be translated to humans by means of robust clinical trials. © 2020 by the Society for Experimental Biology and Medicine.