High-intensity focused ultrasound (HIFU), a noninvasive ablation therapy that has been widely used clinically in ablation of solid tumors, induces immune sensitization. We therefore in this study investigated whether HIFU treatment could enhance the efficacy of a herpes simplex virus 2 (HSV-2) vaccine. First, we observed that in HSV-2-positive cervical intraepithelial neoplasia (CIN) II patients, HIFU treatment induced significantly higher anti-HSV-2 neutralization response than surgical removal. Next, we tested the efficacy of HIFU-treated, UV-inactivated HSV-2-infected cells as a proof-of-concept vaccine in mice. Our data showed that HIFU-treated formulation significantly enhanced HSV-2 antibody titers and neutralization titers, compared to UV-, microwave (MW)-, or freeze-thaw (FT)-treated formulations. HIFU treatment also promoted the Th1/2 cell-mediated response. A long-term full protection was observed in mice that received the HIFU-treated formulation, and no weight loss was detected. Our findings indicate that the novel application of HIFU in vaccine production may represent a rational way to improve vaccine efficacy.IMPORTANCE High-intensity focused ultrasound (HIFU) is mainly used in tumor ablation and tumor vaccinology study. It has been shown to induce immune sensitization and enhance tumor responsiveness to other therapies. Our study has shown enhanced anti-HSV-2 response in HIFU-treated CIN II patients. Furthermore, in a murine model, we have demonstrated that HIFU-treated HSV-2 vaccine induced long-term protective immunity against lethal challenge. Our findings indicate that the novel application of HIFU in vaccine production may represent a rational way to improve vaccine efficacy.