Cerebral ischemia induces massive neuroinflammation and microglial activation. Activated microglia change their phenotype, including metabolism. Inducible nitric oxide synthase (iNOS) and arginase-1 (Arg1) are expressed in activated microglia, and they competitively metabolize l-arginine to NO or precursor of polyamines, respectively. We have reported that a class 4 semaphorin (Sema4D) deficiency inhibits microglial iNOS expression and NO production and promotes microglial proliferation in cerebral ischemia, although underlying mechanisms remain unclear. In this study, we found that Sema4D deficiency alters the balance of competition between iNOS and Arg1, and pushes from NO-production to polyamines-production in microglia. Furthermore, polyamine putrescine promoted proliferation of cultured microglia. Our findings indicate that Sema4D regulates microglial proliferation via l-arginine metabolic pathway in the ischemic brain.