There is a constant need for improved adjuvants to augment the induction of immune responses against tumor-associated antigens (TAA) during immunotherapy. Previous studies have established that listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, exhibits multifaceted effects to boost the stimulation of immune responses to a variety of antigens. However, the direct ability of LLO as an adjuvant and whether it acts as a pathogen-associated molecular pattern (PAMP) have not been demonstrated. In this paper, we show that a detoxified, nonhemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and may activate innate and cellular immune responses by acting as a PAMP. Our investigation of the adjuvant activity demonstrates that dtLLO, either fused to or administered as a mixture with a human papillomavirus type 16 (HPV-16) E7 recombinant protein, can augment antitumor immune responses and facilitate tumor eradication. Further mechanistic studies using bone marrow-derived dendritic cells suggest that dtLLO acts as a PAMP by stimulating production of proinflammatory cytokines and inducing maturation of antigen-presenting cells (APC). We propose that dtLLO is an effective adjuvant for tumor immunotherapy, and likely for other therapeutic settings.