In our previous study, we reported that luteolin might exert neuroprotective functions by inhibiting the production of proinflammatory mediators, thereby suppressing microglial activation. In this study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the effect of ubiquitin-specific processing protease 8 (USP8) in luteolin-treated microglia. Western blot analysis verified that USP8 expression is upregulated by luteolin. Researchers have found that USP8 markedly enhanced the stability of neuregulin receptor degradation protein-1 (Nrdp1), which in turn inhibited the production of proinflammatory cytokines in toll-like receptor-triggered macrophages. We next hypothesized that luteolin inhibits microglial inflammation by regulating USP8 gene expression. After transfecting BV2-immortalized murine microglial cells with USP8, a significant reduction in the degradation of Nrdp1 was observed. USP8 overexpression also reduced the production of lipopolysaccharide (LPS)-induced proinflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). We also found that USP8 siRNA blocked luteolin inhibition of pro-inflammatory gene expression such as iNOS, NO, COX-2, and PGE2. Taken together, our findings suggested that luteolin inhibits microglial inflammation by enhancing USP8 protein production. We concluded that in addition to anti-inflammatory luteolin, USP8 might represent a novel mechanism for the treatment of neuroinflammation and neurodegeneration.