To observe the vaginal microenvironment, and the expression and clinical significance of peripheral blood T lymphocyte subsets, helper T cell 1/helper T cell 2 (Th1/Th2) cytokine of women with cervical high-risk human papillomavirus (HR-HPV) infection. Methods: From February 2020 to February 2021, 250 women who had definite diagnosed as HR-HPV infection were collected, which included 50 women with chronic cervicitis in group A, 150 women with cervical intraepithelial neoplasia (CIN) in group B (50 women with CIN Ⅰgrade in group B1, 50 women with CIN II grade in group B2, and 50 women with CIN III grade in group B3), and 50 women with cervical cancer in group C. Another 50 healthy women were selected in group D during the same time. The vaginal microenvironment status, such as vaginal pH, the status of vaginal cleanliness, trichomoniasis, mold, clue cells, lactobacillus, the values of peripheral blood T lymphocyte subsets (CD4+, CD8+), and the levels of Th1/Th2 cytokines, such as interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor α (TNF-α), and γ-interferon (IFN-γ) of the women in these groups were detected. Results: The rates of vaginal Ph>4.5, abnormal cleanliness, and negative lactobacillus of the women in group A, group B, and group C were significantly higher than those of the women in group D, and which of the women in group B2, group B3, and group C were significantly higher than those of the women in group A and group B1 (all P＜0.05). The values of CD4+ and CD4+/CD8+ of the women in group A, group B, group C, and group D had decreased gradually (P<0.05). The serum levels of IL-2, IFN-γ, TNF-α, IL-4, IL-6, and IL-10 of the women in group A and group B1 were significantly higher than those of the women in group D, while the IFN-γ/IL-4 ratio of the women in group A and group B1 was significantly lower (all P<0.05). The levels of serum IL-2, IFN-γ, TNF-α, and IFN-γ/IL-4 of the women in group A, group B1, group B2, group B3, and group C had decreased gradually with the aggravation of cervical lesions, while the levels of serum IL -4, IL-6 ,and IL-10 of the women of the women in group A, group B1, group B2, group B3, and group C had increased gradually (all P<0.05). Conclusion: The women with HR-HPV infection have the situation of vaginal microenvironmental changes, and T lymphocyte subsets and Th1/Th2 cytokine disorders. The abnormal vaginal microenvironment, the decrease of immune function, and the dominant expression of Th2 of these women may increase the risks of persistent HR-HPV infection and cervical lesions development.