The aim of this study is to investigate the role and mechanism of microglial NOX2 activation in minimally toxic dose of LPS and Syn-elicited synergistic dopaminergic neurodegeneration. NOX2(+/+) and NOX2(-/-) mice and multiple primary cultures were treated with LPS and/or Syn in vivo and in vitro. Neuronal function and morphology were evaluated by uptake of related neurotransmitter and immunostaining with specific antibody. Levels of superoxide, intracellular reactive oxygen species, mRNA and protein of relevant molecules, and dopamine were detected. LPS and Syn synergistically induce selective and progressive dopaminergic neurodegeneration. Microglia are functionally and morphologically activated, contributing to synergistic dopaminergic neurotoxicity elicited by LPS and Syn. NOX2(-/-) mice are more resistant to synergistic neurotoxicity than NOX2(+/+)mice in vivo and in vitro, and NOX2 inhibitor protects against synergistic neurotoxicity through decreasing microglial superoxide production, illustrating a critical role of microglial NOX2. Microglial NOX2 is activated by LPS and Syn as mRNA and protein levels of NOX2 subunits P47 and gp91 are enhanced. Molecules relevant to microglial NOX2 activation include PKC-sigma, P38, ERK1/2, JNK, and NF-DeB(P50) as their mRNA and protein levels are elevated after treatment with LPS and Syn. Combination of exogenous and endogenous environmental factors with minimally toxic dose synergistically propagates dopaminergic neurodegeneration through activating microglial NOX2 and relevant signaling molecules, casting a new light for PD pathogenesis.