Macrophages have increasingly gained interest as a therapeutic target since they represent an integral component of the tumor microenvironment. In fact, M2 macrophage accumulation in solid tumors is associated with poor prognosis and therapy failure. Therefore, reprogramming M2 macrophages towards an M1 phenotype with anti-tumor activity by gene therapy represents a promising therapeutic approach. Herein, we describe recombinant Saccharomyces cerevisiae as a novel gene delivery vehicle for primary human macrophages. Opsonized S. cerevisiae was taken up efficiently by M2 macrophages and initiated the expression of pro-inflammatory cytokines. Recombinant yeast delivered functional nucleic acids to macrophages, especially when constitutively biosynthesized mRNA was used as cargo. Interestingly, expression of the protein encoded for by the delivered nucleic acid was higher in M2 cells when compared to M1 macrophages. Finally, the delivery of mRNA coding for the pro-inflammatory regulators MYD88 and TNF to M2 macrophages induced a prolonged upregulation of pro-inflammatory and cytotoxic cytokines in these cells, suggesting their successful re-education towards an anti-tumor M1 phenotype. Our results suggest the use of yeast-based gene delivery as a promising approach for the treatment of pathologic conditions that may benefit from the presence of M1-polarized macrophages, such as cancer.