Kidney ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) with a poor prognosis and high mortality rate. Recent studies have reported that chrysophanol may have a renal protective effect, but its specific impact and mechanism on IRI remain unclear. This study aimed to explore the effects and mechanisms of chrysophanol on AKI induced by IRI. By utilizing a unilateral kidney IRI mouse model, histopathological changes in the kidney, serum levels of creatinine and urea nitrogen, and protein expressions of apoptosis and mitophagy in kidney tissue were examined. Additionally, a hypoxia/reoxygenation (H/R) model of human kidney-2 (HK-2) cells was established to measure mitochondrial membrane potential levels and reactive oxygen species (ROS). Functional enrichment analysis was performed to screen relevant targets of chrysophanol and AKI, and to verify key targets and pathways. The animal experiments conducted in this study were ethically approved by the Experimental Animal Ethics Committee of Peking University (No. LA2021503). The findings indicate that the IRI group exhibited elevated levels of creatinine and urea nitrogen in serum, significant renal tissue damage, and increased expression of renal injury markers (KIM1), apoptosis-related proteins (cleaved-caspase 3, caspase 3, cytochrome C), and mitochondrial autophagy protein (PINK1) compared to the sham surgery group. Chrysophanol treatment ameliorated the aforementioned pathological changes in a dose-dependent manner in an IRI model. Additionally, it exhibited significant improvements in mitochondrial membrane potential and inhibition of ROS production in HK-2 cells subjected to H/R conditions. Through network pharmacological analysis, HSP90AA1 and PIK3R1 were identified as key targets primarily enriched in the phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) pathway. Real-time quantitative PCR (qPCR) validation confirmed that chrysophanol significantly decreased HSP90AA1 and PIK3R1 mRNA levels in HK-2 cells under H/R conditions, while also enhancing the protein expressions of p-PI3K, PI3K, p-Akt, and Akt. In conclusion, chrysophanol has the potential to enhance AKI by selectively modulating HSP90AA1 and PIK3R1, activating the PI3K/Akt pathway, decreasing apoptosis, regulating mitochondrial autophagy, enhancing mitochondrial membrane potential, and suppressing ROS production. These findings suggest that chrysophanol could serve as a promising therapeutic option for the treatment of AKI.