Recent evidence has suggested that microglial activation plays an important role in the pathogenesis of depression. Activated microglia can secrete various pro-inflammatory cytokines, which may contribute to the development and maintenance of depression. Thus, inhibition of microglial activation may have a therapeutic benefit in the treatment of depression. In the present study, we found that fluoxetine significantly inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α), interleukin- 6 (IL-6) and nitric oxide (NO) and reduced the phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1) and nuclear factor-kappa B (NF-κB) p65 nuclear translocation in microglia. We further found that fluoxetine increased the expression of β-arrestin 2 and enhanced the association of β-arrestin 2 with TAK1-binding protein 1 (TAB1) and disrupted TAK1-TAB1 interaction. Moreover, β-arrestin 2 knock-down abolished the anti-inflammatory effects of fluoxetine in lipopolysaccharide-stimulated microglial cells. Collectively, our findings suggest that β-arrestin 2 is necessary for the anti-inflammatory effects of fluoxetine and offers novel drug targets in the convergent fluoxetine/β-arrestin 2 and inflammatory pathways for treating microglial inflammatory neuropathologies like depression.