Intravesical Bacillus Calmette-Guerin (BCG) administration is the most effective immunotherapy for bladder cancer, but live BCG bacteria may cause serious adverse events and are limited to local administration. Here, we report the formulation of a less toxic treatment using trehalose 6,6′-dimycolate (TDM), a predominant BCG cell wall glycolipid, that activates innate immunity and carries an adjuvant effect. We previously established hydrophilic cationic liposomes incorporating TDM (Lip-TDM) and found that local administration of Lip-TDM exerts antitumor effects on subcutaneously inoculated tumors by inducing CD8⁺ T cell activation via dendritic cell maturation. Here, we further demonstrate that intraperitoneal administration of Lip-TDM exerts antitumor effects in an N-butyl-N-(4-hydroxybutyl) nitrosamine-induced, orthotopic bladder carcinogenesis mouse model. The incidence of bladder cancer in the Lip-TDM treated group was significantly lower compared to the Lip-CON treated group (60.8% [14 out of 23] versus 95.4% [21 out of 22], p < 0.01). Intraperitoneal administration of Lip-TDM enhanced the systemic activation of dendric cells and macrophages, increasing infiltration of CD8⁺ T cells. Furthermore, Lip-TDM treatment induced both natural killer (NK) cell cytotoxic activity against tumors while enriching regulatory NK populations in lymphoid tissues. This antitumor effect was seen in WT mice but not NK cell-depleted mice. These results highlight NK1.1⁺ lymphocytes as essential for Lip-TDM to induce the acquired immune anti-tumor response. Our findings suggest that Lip-TDM is a non-infectious, NK-activating, anti-tumor agent which can be administered intraperitoneally or systemically as a potential alternative to BCG.