In cervical cancer screening, cytology is used as a triage test to refer high-risk human papillomavirus (HR-HPV)-positive women for colposcopy, but its accuracy is inadequate. The present study aimed to demonstrate that the presence of atypical cells with large vacuoles in the cytoplasm of parabasal cells, referred to as vacuolated parabasal cells (VPCs), observed in the Pap smears of HPV-positive women is associated with specific HPV genotypes. Among 2175 patients, 310 with a single HR-HPV infection and cytological diagnosis of high-grade squamous intraepithelial lesions (HSIL) or atypical squamous cells not excluding HSIL (ASC-H) were included, of which 86 were infected with HPV16. Biopsy results revealed that 69 (80.2%) patients had cervical intraepithelial neoplasia Grade 2 or higher (CIN2+). VPCs were found in 47 (54.7%) of HPV16-infected cases, indicating a significant increase of VPCs in HPV16-infected cases ( p < 0.01). Episomal HPV16 load was quantified in 142 VPCs and 156 HSIL (ASC-H) cells using LBC samples from five patients, with a median of 987 copies in VPCs, significantly higher than those in HSIL (ASC-H) cells (176 copies; p < 0.001). VPCs in Pap specimens were identified not only as cells altered by HPV16 infection but also as CIN2+-derived cells and HPV16-producing cells.