Background/Aim: Macrophages are important components of human defense systems and consequently key to antitumor immunity. Human-serum macrophage activation factor (serum MAF) can activate macrophages, making it a promising reagent for anticancer therapy. Materials and Methods: We established four different macrophage subtypes through introduction of different culture conditions to THP-1-and U937-derived macrophages. We assessed phagocytic activity to understand subtype responses to typical macrophage activation factors (MAFs) and the activation mechanisms of serum MAF. Results: All four macrophage subtypes differed in their response to all MAFs. Moreover, serum MAF had two different activation mechanisms: N-acetylgalactosamine (GalNAc)-dependent and GalNAc-independent. Conclusion: Macrophage activation states and mechanisms are heterogeneous.