Purpose To determine the optimal time interval of repeated intravenous injections of iodixanol in rat model and to identify the injury location and causes of renal damage in vivo. Materials and Methods Rats were randomly divided into Control group, Group 1 with one iodixanol injection, and Group 2 with two iodixanol injections. Group 2 was subdivided into 3 cohorts according to the interval between the first and second iodixanol injections as 1, 3, and 5 days, respectively. Blood oxygen level-dependent (BOLD) imaging and diffusion weighted imaging (DWI) were performed at 1 hour, 1 day, 3 days, 5 days, and 10 days after the application of solutions. Results Compared with Group 1 (7.2%), Group 2 produced a remarkable R2⁎ increment at the inner stripe of the renal outer medulla by 15.37% (P = 0.012), 14.83% (P = 0.046), and 13.53% (P > 0.05), respectively, at 1 hour after repeated injection of iodixanol. The severity of BOLD MRI to detect renal hypoxia was consistent with the expression of HIF-1α and R2⁎ was well correlated with HIF-1α expression (r = 0.704). The acute tubular injury was associated with urinary NGAL and increased significantly at 1 day. Conclusions Repetitive injection of iodixanol within a short time window can induce acute kidney injury, the impact of which on renal damage in rats disappears gradually 3–5 days after the injections.