Acute kidney injury (AKI) has been linked to an increased risk of developing chronic kidney disease (CKD). In the pathophysiology of AKI, oxidative stress plays a pivotal role. Previously, we reported that vanin-1, which is involved in oxidative stress, is associated with renal tubular injury. This study aims to determine whether urinary vanin-1 is a biomarker for early diagnosis of AKI in two experimental models: in vivo and in vitro. In a rat model of AKI induced by ischemia/reperfusion (I/R), ischemic AKI was induced in uninephrectomized rats by clamping the left renal artery for 45 min and then reperfusing the kidney. After 1 day of the treatment, urinary N-acetyl-β-D-glucosaminidase (NAG) exhibited a significant increase, but decreased on Day 2 in I/R rats. Serum creatinine (SCr) in I/R rats showed higher than sham-operated rats, but did not reach significance. In contrast, urinary vanin-1 significantly increased on Day 1 and remained significant high level on Day 2 in IR rats. Renal vanin-1 protein was decreased on Days 1 and 3. In line with these findings, immunofluorescence staining demonstrated that vanin-1 was attenuated in the renal proximal tubules in I/R rats. In vitro, the supernatant from HK-2 cells under hypoxia/reperfusion significantly highly included vanin-1 as well as KIM-1 and NGAL. In conclusion, our results suggest that urinary vanin-1 might be a potential novel biomarker for AKI induced by I/R.