Abstract Factors that differentiate risk of cervical cancer associated with infection with single versus multiple HPV types are yet undefined. We hypothesize that E6 oncoprotein is one determining factor. This cross-sectional, multicenter study was performed between 2013 and 2017. A total of 1,781 women were recruited from six hospitals. Samples were tested for presence of 14 types of high-risk HPV DNA. HPV16/18-positive samples were also tested for HPV16/18-E6 oncoprotein. Of 1,781 subjects, 687 (38.6%) tested positive for HPV16/18. HPV16/18 single infections were associated with higher E6 positivity rates compared with multiple infections only for cancer cases (HPV16: 92.2% vs. 76.5%; HPV18: 93.9% vs. 62.1%) but not for normal histopathology or cervical intraepithelial neoplasia. In HPV16/18 coinfection subjects, the positivity rate was 42.9% for HPV16-E6 and 42.9% for HPV18-E6. The combined positivity rate of either HPV16-E6 or HPV18-E6 among HPV16/18 coinfection subjects was 78.6%, similar with HPV16 (74.8%) and HPV18 (79.5%) single-infection subjects. The positivity rates of HPV16/18 E6 oncoprotein varied depending on the HPV-type composition in multiple infection (“clusters”) including HPV types other than 16 and 18. Multiple infection clusters most likely to express HPV16-E6 and HPV18-E6 were HPV16/52 (61.5%) and HPV18/52 (66.7%), and the less were HPV16/45 (10.0%) and HPV18/51 (16.7%), respectively. Patterns of E6 oncoprotein expression varied depending on clustering types. However, expression was greatest in women with single HPV-type infections compared with those with multiple HPV types regardless of histopathology. Our findings provided new insight of natural history of cervical cancer.