OBJECTIVE: To evaluate the efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in the treatment of high-grade squamous intraepithelial lesions of the cervix (HSIL). METHODS: This retrospective study included 22 female patients with histologically confirmed HSIL and high-risk human papillomavirus (HR-HPV) infections. Patients were treated with ALA-PDT once a week for a total of 6 times. All patients had a follow-up period of 3 months and 6 months. The assessment of effectiveness of ALA-PDT was performed by ThinPrep cytology test (TCT), HPV DNA assay, HPV E6/E7 mRNA examination, colposcopy, biopsy, and immunohistochemistry detection. RESULTS: Three months after 5-ALA PDT, the histologic disappearance rate was 81.82% (18/22), while the HPV clearance rate was 54.55% (12/22). At the 6 months checkpoint, the HSIL disappearance rate was 90.91% (20/22) and the HPV clearance rate was 86.36% (19/22). Before PDT, 68.18% of patients (15/22) were confirmed with TCT abnormalities, while only one patient (1/22, 4.55%) was abnormal in the TCT test at 6 months checkpoint. All participants were found HPV E6/E7 mRNA positive initially, while the HPV E6/E7 mRNA negative rate was 90.91% (20/22) at 6 months checkpoint. Additionally, we found a significant difference of the expression of CD4+ and CD8+ T cells and HPV E6 and E7 proteins before ALA-PDT and at 3 months follow-up (P < 0.01). No severe side effects were seen. CONCLUSIONS: Topical 5-ALA PDT is an effective treatment for cervical HSIL with HR-HPV infection.