Acute kidney injury (AKI) is mainly caused by ischemia-reperfusion (I/R), with high clinical mortality since there is a lack of definite and effective drug intervention except for renal replacement therapy. Previous research emphasized that ferroptosis is the pivotal process in I/R injury of renal. Ginsenoside Rb1 is a chemical compound belonging to the ginsenoside family and has been proved to have benefits for kidney diseases, but its specific mechanism in AKI is uncovered. Here we discovered Rb1 could mitigate ferroptosis in renal tubular epithelial cells of AKI animals. Mechanically, Rb1 significantly inhibited oxidative stress including promotion of GSH, reduction of malondialdehyde (MDA) and reactive oxygen species (ROS), also reduced inflammatory response, then relieved renal injury in I/R mice. RSL3, the inhibitor of GPX4, or Erastin, the inhibitor of SLC7A11, could induce ferroptosis and eliminate the protective effect of Rb1. Also, a ferroptosis inhibitor or ROS scavenger could simulate the protective effect of Rb1 on renal tubular epithelial cells. In conclusion, we confirmed that Rb1 promotes GSH synthesis by preventing oxidative stress, upregulating the expression of GPX4 and SLC7A11, and ultimately scavengers of ROS or MDA, and reduces the occurrence of ferroptosis in renal tubular epithelial cells. Rb1 has potential benefits for AKI patients.