Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor-associated macrophages (TAM) have a string of immunosuppressive functions and they are considered a therapeutic target in cancer. This study aimed to analyze the effects of trabectedin, an antitumor agent, on the tumor-microenvironment through the characterization of the electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages. Trabectedin does not directly interact with K_V1.5 and K_V1.3 channels, but their treatment (16 h) with sub-cytotoxic concentrations of trabectedin increased their K_V current due to an upregulation of K_V1.3 channels. In vitro generated TAM (TAM_iv) exhibited an M2-like phenotype. TAM_iv generated a small K_V current and express high levels of M2 markers. K⁺ current from TAMs isolated from tumors generated in mice is a mixture of K_V and K_Ca, and in TAM isolated from tumors generated in trabectedin-treated mice, the current is mostly driven by K_Ca. We conclude that the antitumor capacity of trabectedin is not only due to its effects on tumor cells, but also to the modulation of the tumor microenvironment, due, at least in part, to the modulation of the expression of different macrophage ion channels.