The Mycobacterium bovis Bacille Calmett-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of μm, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166 nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor cells (MBT-2) in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with in naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug.