Clozapine is an atypical antipsychotic agent used in the treatment of schizophrenia and severe mood disorders. Accumulating evidence suggests that neuroinflammation is closely associated with the pathogenesis of various neurodegenerative diseases and psychiatric disorders. Clozapine exerts anti-inflammatory activity. However, the molecular mechanism underlying the anti-inflammatory activity of clozapine is poorly understood. In this study, we found that clozapine suppressed lipopolysaccharide (LPS)-induced phosphorylation of IκBα at Ser-32 and of p65/RelA at Ser-468, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-dependent transcriptional activity in microglial cells. Clozapine downregulated LPS-induced Akt phosphorylation at Ser-473. Pharmacological Akt inhibitors ameliorated LPS-induced NF-κB activation. Removal of extracellular Ca2+ by EGTA or sequestration of intracellular Ca2+ by BAPTA-AM attenuated LPS-induced Akt phosphorylation. Treatment with calmodulin (CaM) antagonists and the CaM kinase inhibitor, KN-93, also prevented LPS-induced Akt and NF-κB activation, suggesting that Ca2+/CaM-dependent Akt activation is critical in LPS-induced NF-κB activation in microglia. These results suggest that clozapine exhibits anti-inflammatory activity through the inhibition of Ca2+/CaM/Akt-mediated NF-κB activation.