Antimicrobial peptides (AMPs) are components of the innate immune system and form the first defense against pathogens for various organisms. In the present study, we assessed whether CSP32, a novel AMP oligomer of bacitracin isolated from a strain of Bacillus spp., regulates the polarization of murine macrophage-like RAW 264.7 cells. CSP32 stimulated phagocytosis while inducing the appearance of the typical M1 polarized macrophage phenotype; these M1 macrophages play a role in host defense against pathogens. Furthermore, our results showed that CSP32 enhanced the expression and production of pro-inflammatory mediators, such as cytokines and chemokines. In addition, the CSP32-stimulated inflammatory mediators were induced mainly by the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) signaling pathway during M1 macrophage polarization. In particular, CSP32 markedly increased the numbers of Ca2+-positive macrophages while upregulating phospholipase C and activating protein kinase Cε. Furthermore, the inhibition of intracellular Ca2+ by BAPTA-AM, a Ca2+ chelator, significantly suppressed the CSP32-mediated phagocytosis, inflammatory mediator production, and NF-κB activation. In conclusion, our data suggested that CSP32-stimulated M1 macrophage polarization is dependent on the calcium signaling pathway and may result in enhanced immune capacities.