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This study aims to evaluate the therapeutic potential of cationic liposomal neostigmine bromide (NB), a novel drug delivery system, for the treatment of detrusor underactivity. By comparing the characteristics of NB‐liposomes (NLP), NB‐β‐cyclodextrin inclusion complex liposomes (NCLP), and NB‐mesoporous silica nanoparticle@CaCO3 liposomes (NMCLP), NMCLP is selected as the main research subject. It has an average particle size and zeta potential of 100 nm and +50 mV, and its encapsulation efficiency and loading capacity of NB are 14.75% and 12.8%, respectively. Most importantly, NMCLP shows the best in vitro release performance among the three liposomes, demonstrating its ability in sustained release of NB. During cell and animal assays, efficient cellular uptake of liposomes through liposome‐specific pathways is observed, facilitating targeted drug delivery, and in vivo experiments demonstrate the efficacy of NMCLP in improving bladder function in mice. Urodynamic measurements show increased bladder capacity and reduced voiding pressure, indicating enhanced bladder muscle activity. Histological analysis reveals the distribution and deep penetration of NMCLP within bladder tissues, supporting its localized drug effect. Therefore, NMCLP holds promise as a targeted and effective therapeutic strategy for detrusor underactivity. [ABSTRACT FROM AUTHOR]