Many cancer vaccines are not successful in clinical trials, mainly due to the challenges associated with breaking immune tolerance. Herein, we report a new strategy using an adjuvant-protein-antigen (three-in-one protein conjugates with built-in adjuvant) as an anticancer vaccine, in which both the adjuvant (small-molecule TLR7 agonist) and tumor-associated antigen (mucin 1, MUC1) are covalently conjugated to the same carrier protein (BSA). It is shown that the protein conjugates with built-in adjuvant can increase adjuvant’s stimulation, prevent adjuvant’s systemic toxicities, facilitate the codelivery of adjuvants and antigens, and enhance humoral and cellular immune responses. The IgG antibody titers elicited by the self-adjuvanting three-in-one protein conjugates were significantly higher than those elicited by the vaccine mixed with TLR7 agonist (more than 15-fold) or other traditional adjuvants. Importantly, the potent immune responses against cancer cells suggest that this new vaccine construct is an effective strategy for the personalized antitumor immunotherapy.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.