Background: polymorphonuclear neutrophils (PMNs) are main effector cells in the inflammatory responses. The significance of PMN infiltration in tumor microenvironment remains to be clarified. Metastatic melanoma is the most lethal type of skin cancer with an increasing incidence over the last decades. Few studies investigated the role of PMNs in human melanoma. The aim of this study was to investigate the role of PMNs and their mediators in human melanoma; (2) Methods: highly purified human PMNs from healthy donors were stimulated, in vitro, with conditioned media derived from the melanoma cell lines SKMEL28 and A375 (melanoma-CM) as well as from primary melanocytes as control. PMN functions (chemotaxis, survival, activation, cell tracking, morphology and NETs release) were evaluated; (3) Results: we found that the A375 cell line produced soluble factors able to promote PMN chemotaxis, survival, activation, and to modify PMN morphological changes and kinetic properties. Furthermore, both melanoma cell lines CM induced activation and the release of neutrophil extracellular traps (NETs) from PMNs. By contrast, primary melanocytes CM did not modify any PMN biological behavior. In addition, serum levels of myeloperoxidase (MPO), matrix metalloprotease-9 (MMP-9), CXCL8/IL-8, granulocyte and monocyte colony stimulating factor (GM-CSF) and NETs components were significantly increased in advanced melanoma patients compared to healthy controls; (4) Conclusions: melanoma cell lines produce soluble factors able to ‘educate’ PMNs towards an activated functional state. Metastatic melanoma patients display increased circulating levels of neutrophil-related mediators and NETs, suggesting that a neutrophil-related signature exists in metastatic melanoma patients. Further investigations are needed to better understand the role of these “tumor-educated neutrophils” in modifying melanoma cell behavior.