Interactions between Sema4D and its receptors, PlexinB1 and CD72, induce various functions, including axon guidance, angiogenesis, and immune activation. Our previous study revealed that Sema4D is involved in the upregulation of nitric oxide production in microglia after cerebral ischemia. In this study, we investigated the underlying mechanisms of the enhancement of microglial nitric oxide production by Sema4D. Primary microglia expressed PlexinB1 and CD72, and cortical microglia expressed CD72. Sema4D promoted nitric oxide production and slightly inhibited Erk1/2 phosphorylation in microglia. Partial Erk1/2 inhibition enhanced microglial nitric oxide production. Inhibition of Erk1/2 phosphorylation induced the expression of Ifn-beta mRNA, and IFN-beta promoted nitric oxide production in microglia. In the ischemic cortex, the expression of Ifn-beta mRNA was downregulated by Sema4D deficiency. These findings indicated that the enhancement of nitric oxide production by Sema4D is involved in partial Erk1/2 inhibition and upregulation of IFN-beta. (C) 2019 Published by Elsevier Inc.