BACKGROUND: Macrophages, with many different phenotypes play a major role during wound healing process, secreting the cytokines crucial to angiogenesis, cell recruitment and ECM remodeling. Therefore, macrophage-derived cytokines may be attractive therapeutic resource for wound healing. METHODS: To obtain a conditioned media (CM) from macrophages, human monocyte THP-1 cells were seeded on TCP or human fibroblast-derived matrix (hFDM) and they were differentiated into M1 or M2 phenotype using distinct protocols. A combination of different substrates and macrophage phenotypes produced M1- and M2-CM or M1-hFDM- and M2-hFDM-CM, respectively. Proteome microarray determines the cytokine contents in those CMs. CMs-treated human dermal fibroblast (hDFB) was analyzed using collagen synthesis and wound scratch assay. Concentrated form of the CM (CCM), obtained by high-speed centrifugation, was administered to a murine full-thickness wound model using alginate patch, where alginate patch was incubated in the M2-CCM overnight at 4 °C before transplantation. On 14 day post-treatment, examination was carried out through H&E and Herovici staining. Keratinocyte and M2 macrophages were also evaluated via immunofluorescence staining. RESULTS: Cytokine analysis of CMs found CCL1, CCL5, and G-CSF, where CCL5 is more dominant. We found increased collagen synthesis and faster wound closure in hDFB treated with M2-CM. Full-thickness wounds treated by M2-hFDM-CCM containing alginate patch showed early wound closure, larger blood vessels, increased mature collagen deposition, enhanced keratinocyte maturation and more M2-macrophage population. CONCLUSION Our study demonstrated therapeutic potential of the CM derived from M2 macrophages, where the cytokines in the CM may have played an active role for enhanced wound healing.