The pathogenesis of kidney damage involves complicated interactions between vascular endothelial and tubular cell destruction. Evidence has shown that vitamin D may have anti-inflammatory effects in several models of kidney damage. In this study, we evaluated the effects of synthetic vitamin D on levofloxacin-induced renal injury in rats. Forty-two white Albino rats were divided into six groups, with each group comprising seven rats. Group I served as the control (negative control) and received intraperitoneal injections of normal saline (0.5 ml) once daily for twenty-one days. Group II and Group III were treated with a single intraperitoneal dose of Levofloxacin (50 mg/kg/day) and (100 mg/kg/day), respectively, for 14 days (positive control groups). Group IV served as an additional negative control and received oral administration of vitamin D3 (500 IU/rat/day) for twenty-one days. In Group V, rats were orally administered vitamin D3 (500 IU/rat/day) for twenty-one days, and intraperitoneal injections of Levofloxacin (50 mg/kg/day) were administered on day 8 for 14 days. Group VI received oral vitamin D3 supplementation (500 IU/rat/day) for twenty-one days, followed by intraperitoneal injections of Levofloxacin (100 mg/kg/day) on day 8 for fourteen days. Blood samples were collected to measure creatinine, urea, malondialdehyde, glutathione reductase, and superoxide dismutase levels. Compared to the positive control group, vitamin D supplementation lowered creatinine, urea, and malondialdehyde levels, while increasing glutathione reductase and superoxide dismutase levels. Urea, creatinine, and malondialdehyde levels were significantly (p<0.05) higher in rats administered LFX 50mg and 100mg compared to rats given (LFX + vitamin D). The main findings of this study show that vitamin D reduces renal dysfunction, suggesting that vitamin D has antioxidant properties and may be used to prevent renal injury.