Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) and is characterized by widespread tubular and microvascular damage. The tumor suppressor p53 is up-regulated after IRI and contributes to renal injury in part by promoting apoptosis. Acute, short-term inhibition of p53 with pifithrin conveys significant protection after IRI. The HIF-1 pathway is also activated after IRI and has opposing effects to those promoted by p53. The balance between the HIF-1 and p53 responses can determine the outcome of IRI. In this manuscript, we investigate whether p53 regulates the HIF-1 pathway in a rodent model of IRI. HIF-1 is principally expressed in the collecting tubules (CT) and thick ascending limbs (TAL) under physiologic conditions. However, inhibition of p53 with pifithrin increases the faint expression of HIF-1 in proximal tubules (PT) under physiologic conditions. Twenty-four hours after IRI, HIF-1 expression is decreased in both CT and TAL. HIF-1 expression in the PT is not significantly altered after IRI. Acute inhibition of p53 significantly increases HIF-1 expression in the PT after IRI. Additionally, pifithrin prevents the IRI-induced decrease of HIF-1 in the CT and TAL. Parallel changes are observed in the HIF-1 transcriptive target, carbonic anhydrase-9. Finally, inhibition of p53 prevents the dramatic changes in pVHL morphology and expression after IRI. We conclude that activation of p53 after IRI mitigates the concomitant activation of the protective HIF-1 pathway. Modulating the interactions between the p53 and HIF-1 pathway can provide novel options in the treatment of AKI.