Macrophages are important innate immune defense system cells in the fight against bacterial and fungal pathogenic infections. They exhibit significant plasticity, particularly with their ability to undergo functional differentiation. Additionally, HIF1a is critically involved in the functional differentiation of macrophages during inflammation. However, the role of macrophage HIF1a in protecting against different pathogenic infections remains unclear. In this study, we investigated and compared the roles of HIF1a in different macrophage functional effects of bacterial and fungal infections in vitro and in vivo. We found that bacterial and fungal infections produced similar effects on macrophage functional differentiation. HIF1a deficiency inhibited pro-inflammatory macrophage functional activities when cells were stimulated with LPS or curdlan in vitro or when mice were infected with L. monocytogenes or C. albicans in vivo, thus decreasing pro-inflammatory TNFa and IL-6 secretion associated with pathogenic microorganism survival. Alteration of glycolytic pathway activation was required for the functional differentiation of pro-inflammatory macrophages in protecting against bacterial and fungal infections. Thus, the HIF1a-dependent glycolytic pathway is essential for pro-inflammatory macrophage functional differentiation in protecting against bacterial and fungal infections