We reported previously that phagocytosis of apoptotic cells by U937 cells was enhanced by the treatment with oridonin that showed high activity to induce the generation of reactive oxygen species (ROS) in many cells. ROS, important signaling molecules, are involved in the immune defenses, cell repair and proliferation. In this study, oridonin caused modest amount of ROS generation in U937 cells, with hydrogen peroxide (H2O2) and hydroxyl free radical (OH) as the major types. Meanwhile, H2O2 and OH were positive regulators involved in oridonin-enhanced engulfment of apoptotic cells through down-regulating mitochondrial membrane potential (MMP) and inducing autophagy. The ROS-mediated phagocytosis was independent of cellular adenosine triphosphate (ATP) levels. H2O2 and OH generation also activated phosphatidylinositol 3-kinases-Akt (PI3K-Akt) and phospholipase C γ-protein kinase C(PLC γ)-Ras-Raf-ERK signaling pathways, which were essential for oridonin-induced engulfment of apoptotic cells. Phagocytosis, the loss of MMP, autophagy and the activated signaling pathways were all suppressed by ROS scavenger N-acetyl-l-cysteine (NAC), H2O2 scavenger catalase or OH scavenger glutathione (GSH). However, superoxide anion (O2-) and its scavenger superoxide dismutase (SOD) did not significantly affect these oridonin-induced biological processes.