Acute kidney injury (AKI) is caused by hypoxia-reoxygenation (H/R), which is a kidney injury produced by a variety of causes, resulting in the remaining portion of the kidney function being unable to maintain the balance for performing the tasks of waste excretion metabolism, and electrolyte and acid-base balance. Many studies have reported the use of Chinese medicine to slow down the progression and alleviate the complications of chronic renal failure. Chrysophanol is a component of Rheum officinale Baill, a traditional Chinese medicine that has been clinically used to treat renal disease. We aimed to study the nephroprotective effect of chrysophanol on hypoxia/ reoxygenation (H/R)-induced cell damage. The results showed that chrysophanol prevented H/R-induced apoptosis via downregulation of cleaved Caspase-3, p-JNK, and Bax but upregulation of Bcl-2 expression. In contrast, chrysophanol attenuated H/R-induced endoplasmic reticulum (ER) stress via the downregulation of CHOP and p-IRE1α expression. Our data demonstrated that chrysophanol alleviated H/R-induced lipid ROS accumulation and ferroptosis. Therefore, we propose that chrysophanol may have a protective effect against AKI by regulating apoptosis, ER stress, and ferroptosis.