AbstractTumor‐associated macrophages (TAMs) represent the majority of the immune cells present in the tumor microenvironment. These macrophages exhibit an anti‐inflammatory (M2)‐like physiological state and execute immune‐suppressive and tumor‐supporting properties. With TAMs being plastic, there is a growing interest in reprogramming them toward a pro‐inflammatory (M1)‐like phenotype that exhibits anti‐tumoral properties. Recent studies have demonstrated that both engineered vesicles derived from macrophages and endogenous extracellular vesicles produced by macrophages can be programmed to alter macrophage phenotype. Here it is demonstrated that pro‐inflammatory macrophage‐engineered subcellular vesicles (MEVs) have differential properties based on their organelle of origin. Endoplasmic reticulum specific MEVs (erMEVs) treated M2 macrophages exhibit enhanced pro‐inflammatory cytokine production compared to plasma membrane specific MEVs (pmMEVs) treated M2 macrophages. In addition, under in vitro co‐culture conditions, erMEVs elicit superior efficacy in suppressing the viability of cancer cells compared to the same concentration of pmMEVs. Furthermore, erMEVs and pmMEVs maintain differences in their membrane proteins, that regulate the repolarization efficacy of M2 macrophages toward an M1‐like phenotype. In addition, The M2 to M1 repolarizing efficacy of MEVs can be altered by changing the activity of the membrane proteins present on erMEVs or pmMEVs.