The importance of microglial reactive oxygen species (ROS) signaling in neuroinflammatory processes has been well demonstrated; however, relatively little is known regarding the related mechanisms underlying these processes. Here, we show that ROS-dependent signal pathways that govern microglial phagocytosis are highly dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activation. Specifically, phagocytosis was greatly reduced by both antioxidant and Nox inhibitor treatments in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Additionally, there was a marked reduction in intracellular ROS content. These results suggest that Nox is the main ROS source for LPS-induced microglial phagocytosis. More decisive evidence for the involvement of ROS in phagocytosis was obtained from an examination of phosphatidyl inositol 3-kinase (PI3-K) and p38 mitogen-activated protein kinase (MAPK) signal pathway activation under reduced ROS levels. These two kinases were activated by LPS treatment and inhibited by ROS neutralization and Nox inhibition. We conclude that microglial phagocytosis requires ROS-dependent PI3-K and p38 MAPK activation and that Nox-derived ROS functions as an upstream regulator of both PI3-K and p38 MAPK. These findings will provide a fundamental basis for a therapeutic modality in inflammation-mediated neurodiseases.