Abstract Cancer immunotherapies utilize distinct mechanisms to harness the power of the immune system to eradicate cancer cells. Therapeutic vaccines, aimed at inducing active immune responses against an existing cancer, are highly dependent on the immunological microenvironment, where many immune cell types display high levels of plasticity and, depending on the context, promote very different immunologic outcomes. Among them, plasmacytoid dendritic cells (pDC), known to be highly immunogenic upon inflammation, are maintained in a tolerogenic state by the tumor microenvironment. Here, we report that intratumoral (i.t.) injection of established solid tumors with CpG oligonucleotides-B (CpG-B) inhibits tumor growth. Interestingly, control of tumor growth was independent of tumor-associated pDC, which remained refractory to CpG-B stimulation and whose depletion did not alter the efficacy of the treatment. Instead, tumor growth inhibition subsequent to i.t. CpG-B injection depended on the recruitment of neutrophils into the milieu, resulting in the activation of conventional dendritic cells, subsequent increased antitumor T-cell priming in draining lymph nodes, and enhanced effector T-cell infiltration in the tumor microenvironment. These results reinforce the concept that i.t. delivery of TLR9 agonists alters the tumor microenvironment by improving the antitumor activity of both innate and adaptive immune cells. Significance: Intratumoral delivery of CpG-B disrupts the tolerogenic tumor microenvironment and inhibits tumor growth. Cancer Res; 78(12); 3280–92. ©2018 AACR.