Macrophages play a crucial role in inflammation, a defense mechanism of the innate immune system. Metabolic function powered by glucose transporter isoform 1 (Glut1) is necessary for macrophage activity during inflammation. The present study investigated the roles of cystathionine-γ-lyase (CSE) and its byproduct, hydrogen sulfide (H2S), in macrophage glucose metabolism to explore the mechanism by which H2S acts as an inflammatory regulator in lipopolysaccharide- (LPS) induced macrophages. Our results demonstrated that LPS-treated macrophages increased Glut1 expression. LPS-induced Glut1 expression is regulated via nuclear factor (NF)-κB activation and is associated with phosphatidylinositol-3-kinase PI3k activation. Small interfering (si) RNA-mediated silencing of CSE decreased the LPS-induced NF-κB activation and Glut1 expression, suggesting a role for H2S in metabolic function in macrophages during pro-inflammatory response. Confoundingly, treatment with GYY4137, an H2S-donor molecule, also displayed inhibitory effects upon LPS-induced NF-κB activation and Glut1 expression. Moreover, GYY4137 treatment increased Akt activation, suggesting a role in promoting resolution of inflammation. Our study provides evidence that the source of H2S, either endogenous (via CSE) or exogenous (via GYY4137), supports or inhibits the LPS-induced NF-κB activity and Glut1 expression, respectively. Therefore, H2S may influence metabolic programming in immune cells to alter glucose substrate availability that impacts the immune response.