Cancer vaccines are promising to treat malignancy by delivering antigens and adjuvants to elicit host immunity. Beyond aluminum adjuvants, liposomes show efficient adjuvant effects through regulating the accumulation, internalization and release of payloads. However, it remains unknown that whether the liposome will perform intrinsic adjuvant effects in the absence of antigens and adjuvants. Herein, a library of antigen/adjuvant‐free liposomes with variable surface charges has been developed and it has been found that highly anionic liposomes show promising adjuvant effects for boosting immune responses. The anionic liposome mobilizes the MyD88 pathways of dendritic cells (DCs) to activate T helper cells and CD8+ T cells. The anionic liposomes enhance host immunity by regulating the population of Th1, Th2 and regulatory T cells (Tregs), and boost adaptive CD8+ T cells in lymphoid organs with good biosafety. It shows the most efficient protection against MC38 colorectal cancer in mice after a parallel injection of antigens and anionic liposomes. Overall, this study reveals that the surface charge of liposome affects its adjuvant efficiency and provides an anionic nanosized adjuvant formulation for enhancing immunization.