The aim of this survey was to examine the knowledge, attitudes, and behavior of gynecologists in terms of human papillomavirus (HPV) DNA testing as a primary screening tool for cervical cancer. A national cross-sectional web survey was carried out through multistage sampling using an overall sample of 1000 gynecologists. Gynecologists were asked to fill in a self-administered questionnaire exploring their knowledge, attitudes, and practice toward cervical cancer screening and HPV-DNA testing. A total of 582 gynecologists completed the web questionnaire. Of these, 24.5% were uncertain on the higher sensitivity of HPV-DNA compared with the Pap test, whereas 19% were uncertain on the role of the HPV-DNA test as a primary test in women younger than 30 years old and only 44.9% knew that a negative HPV-DNA test allows for an extension of the test interval to 5 years. Most gynecologists showed a definite positive attitude on the role of screening for cervical cancer prevention and were prepared to accept new technologies. The HPV-DNA test was considered highly effective by 86.9%, whereas 94% recommend/perform HPV-DNA tests in women older than 30 years of age; 25.5% performed HPV-DNA as a primary test, followed by a Pap test in those cases that were positive. Only 56.3% recommended/performed HPV-DNA tests 1 year after a positive HPV-DNA test, followed by a negative Pap test, whereas 42.9% recommended colposcopy. Although the use of the HPV-DNA test is very widespread among Italian gynecologists performing cervical cancer screening, there is lack of standardization of practices according to current guidelines.