Proprotein convertases (PCs) are proteases which have an important regulatory function in a wide variety of biological processes. However, these enzymes have a key role in the activation of many cancer-related proteins and promotes the malignant phenotype of cancer cells. Here, with proteomics and biological tests, we investigated the consequences of the use of a peptidomimetic PCs inhibitor on glioma cells and macrophages in the same time and its potential application to cancer therapy. Thus, we demonstrated that the PCs inhibitor acts on macrophage activation, characterized by the secretion of pro-inflammatory and anti-tumoral factor. Also, the PCs inhibitor triggers a strong decrease of the proliferation and invasion of glioma cells, even when they are associated with macrophages in spheroids. In fact, the PCs inhibitor induces great changes in the intracellular and secreted protein profiles of the glioma cells. Many of cancerous processes are affected, such as cell growth, proliferation, angiogenesis and the immunosuppressive capacity of cancer cells. Taken together, these data, establish that the inhibitor acts both on cancer cells and macrophages and can be used as a potential anti-cancer therapeutic strategy.