Catanionic vesicles are considered a potential alternative to liposomes for drug delivery systems because of their greater stability and lower cost. Before using catanionic vesicles in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. Using flow cytometry, we examined the intracellular responses—reactive oxygen species (ROS) content, mitochondrial membrane potential, cell size and complexity, and cell cycle profiles—in U-937 human macrophages treated with positively charged catanionic vesicles. Kinetic hydrogen peroxide production initially increased at lower concentrations (4–10 nM) but declined at higher concentrations (40 nM and 80 nM) over the entire incubation period. Superoxide content generation, however, increased over the entire concentration range and incubation period. Catanionic vesicles decreased mitochondrial membrane potential for every concentration after 4 h of incubation but caused a significant fluctuation in mitochondrial membrane potential at 6 h. After 6 h of incubation, catanionic vesicles produced more changes in cell size and complexity than after 4 h. The increase in the subG1 population of cells treated with catanionic vesicles at higher doses indicated that apoptosis progressed. Positively charged catanionic vesicles induced different activated patterns of ROS generation and changes in mitochondrial membrane potential than did cationic liposomes. The nature of the interactions between macrophages and catanionic vesicles is of great importance for the design of safer and more effective delivery systems for macrophages. Our findings contribute to a better understanding of the molecular action of catanionic vesicles in the cellular system.