Inhibition of the tumor suppressor p53 diminishes tubular cell apoptosis and protects renal function in animal models of acute kidney injury (AKI). Therefore, targeting p53 has become an attractive therapeutic strategy in the approach to AKI. Although the acute protective effects of p53 inhibition in AKI have been examined, there is still relatively little known regarding the impact of acute p53 inhibition on the chronic sequelae of AKI. Consequently, we utilized the p53 inhibitor pifithrin-α to examine the long-term effects of p53 inhibition in a rodent model of ischemic AKI. Male Sprague-Dawley rats were subjected to bilateral renal artery clamping for 30 min followed by reperfusion for up to 8 wk. Pifithrin-α or vehicle control was administered at the time of surgery and then daily for 2 days [brief acute administration (BA)] or 7 days [prolonged acute administration (PA)]. Despite the acute protective effect of pifithrin-α in models of ischemic AKI, we found no protection in the microvascular rarefaction at 4 wk or development fibrosis at 8 wk with pifithrin-α administered on the BA schedule compared with vehicle control-treated animals. Furthermore, pifithrin-α administered on a PA schedule actually produced worse fibrosis compared with vehicle control animals after ischemic injury [21%/area (SD4.4) vs.16%/area (SD3.6)] as well as under sham conditions [2.6%/area (SD1.8) vs. 4.7%/area (SD1.3)]. The development of fibrosis with PA administration was independent of microvascular rarefaction. We identified enhanced extracellular matrix production, epithelial-to-mesenchymal transition, and amplified inflammatory responses as potential contributors to the augmented fibrosis observed with PA administration of pifithrin-α.