Abstract Recombinant adeno-associated virus (rAAV) is being tested as a vaccine vector, but the cellular immune responses elicited in animal tumor models have not been completely protective. The adjuvant effects of the TLR7 agonist, imiquimod, and the TLR9 agonist, ODN1826, were tested with rAAV expressing the melanoma antigen, Trp2. Mice immunized with rAAV-TRP2 and either TLR agonist alone generated T-helper-1 antitumor immune responses. Antitumor activity in all experiments was still incomplete. Furthermore, antitumor activity was not achieved when the combination of ODN1826 and imiquimod was used as adjuvant. In vitro, the combination increased IL-10 production by dendritic cells. In vivo, the combination reduced T-helper-1 response and dendritic cell activation and increased myeloid suppressor cells; regulatory T cells were not significantly modulated. Depletion of myeloid derived suppressor cells enhanced the antitumor activity of immunization with rAAV-TRP2 and the imiquimod-ODN1826 combination; depletion of regulatory T cells did not. TLR7 and TLR9 agonists can be used to enhance the immune response to rAAV immunogens, but antagonism can be observed when combined. Suppressor mechanisms, including those mediated by myeloid cells, may negatively regulate the antitumor immune response.