Background/Aim: Serum-derived macrophage activating factor (serum-MAF) is expected to have adjuvant effects through rapid phagocytic activation, which depends on F-actin accumulation in multi-layered membrane ruffles induced within 5 min after serum-MAF addition. This study aimed to elucidate the importance of annexin A2, which is a multifunctional Ca 2+ -binding protein related to cytoskeletal membrane dynamics, in serum-MAF signalling. Materials and Methods: Annexin A2 and F-actin localizations were analyzed via immunostaining and confocal microscopy. Using EGTA as chelator, the role of Ca 2+ in serum-MAF signalling was examined. Results: Annexin A2 was found to translocate from the cytosol to the cell cortex within 30 s of serum-MAF stimulation. Ca 2+ chelation inhibited the translocation of annexin A2, frill-like structure formation, and phagocytic activation by serum-MAF. Conclusion: Annexin A2 and Ca 2+ were responsible for the rapid phagocytic activation by serum-MAF. This study provides an understanding of phagocytic activation in macrophages, which could be beneficial for cancer immunotherapy.Macrophages play a pivotal role in activating the entire immune system by phagocytosing pathogens (1). Thus, the enhancement of macrophage phagocytic activity is expected to maintain human homeostasis, including cancer immunity (1, 2). We previously described the rapid and strong phagocytic activity of the serum derived macrophage activating factor, serum-MAF (3). Serum-MAF is a human serum treated with galactosidase and sialidase (4). Serum-MAF-activated macrophages form a frill-like structure: Factin accumulates multi-layered membrane ruffles and is responsible for effective phagocytosis within 5 min (5,6). The mechanisms underlying such rapid actin reorganization are intriguing at the cytological and immunological levels.Annexin A2 is one of the actin-binding proteins. It is a multifunctional Ca 2+ -and lipid-binding protein expressed in approximately all human tissues and cell types. The functions of annexin A2 include cytoskeleton-membrane interactions, and participation in a broad range of intracellular processes, such as membrane domain organization, membrane fusion, and vesicle aggregation, which are involved in many cellular functions, such as exocytosis, endocytosis, and phagocytosis (7-11).We hypothesized that the regulation of cytoskeletonmembrane interactions is involved in rapid F-actin reorganization in frill-like structures. This study analyzed the role of endogenous annexin A2 in serum-MAF-activated macrophages. Materials and MethodsCell culture. The THP-1 cell line was obtained from RIKEN BRC (Tokyo, Japan). THP-1 cells were differentiated into macrophages by incubation with 400 ng/ml 12-O-tetradecanoyl-13-acetate (TPA;