In this study, we aimed to develop an efficient drug delivery system by reassembling vacuoles isolated from Saccharomyces cerevisiae. Initially, we assessed the impact of vacuolar enzymes on the efficacy of the loaded antibiotic polymyxin B (PMB), by conducting antibacterial activity tests using Shigella flexneri and Salmonella enteritidis. The results showed that vacuolar enzymes inhibited the effectiveness of PMB, highlighting the limitations of using natural vacuoles as drug carriers. To overcome this, we proposed a new drug delivery system called reassembled vacuoles (ReV). ReV particles were created by removing vacuolar enzymes and reassembling the vacuolar membrane through extrusion. ReV demonstrated improved structural stability, a more uniform size, and enhanced PMB release compared to natural vacuoles. Encapsulation efficiency tests revealed high loading efficiency for both normal vacuoles (NorV) and ReV, with over 80% efficiency at concentrations up to 600 μg/mL. The antibacterial activity of PMB-loaded ReV showed comparable results to PMB alone, indicating the potential of ReV as a drug delivery system. In conclusion, reassembled vacuoles offer a promising approach for drug delivery, addressing the limitations of natural vacuoles and providing opportunities for targeted and efficient drug release.