Introduction: Cervical cancer (CC) is a serious public health concern in Serbia, due to opportunistic screening still being in force, which led to twice higher than the average incidence rate of cervical cancer in Europe. Despite the fact that early detection and treatment services of CC are available at no additional cost, majority of women use inadequate screening services in Serbia. Objective: This study aimed to examine the link between the knowledge about CC and Papanicolaou (Pap) test and perception of barriers to women's participation in CC screening. Materials and Methods: The study included 300 women aged 21 to 69, with a place of residence in the city of Belgrade (Serbia), who were attending for their medical examination to the University Clinic of Gynecology and Obstetrics -"Narodni front", from June through December 2014. A survey instrument to collect data was an adapted questionnaire for the assessment of knowledge about and barriers to CC and Pap test. Patients were divided into three groups: a study group consisted of women attending irregularly (over three years), women who never participated in screening, and a control group that included women regularly participating in screening. Results: Women regularly participating in screening (52.7%) had adequate knowledge about CC and Pap test, while women who irregularly (79.4%) or never participated (71.9%) did not have any adequate knowledge. There was a significant statistical difference between the CC and Pap test awareness in a group of respondents who regularly participated in comparison to respondents who irregularly or never participated in screening (x(2) = 27.772, p = 0.000). Regarding knowledge about human papillomavirus (HPV), 80% of women did not know that Pap test cannot be used for detection of HPV, as well as that abnormal Pap test result may be due to HPV (61.7%). Majority of women (93.7%) had poor knowledge about Pap test role in CC early detection and considered Pap test to be used to diagnose CC. The authors found a significant statistical correlation between participation of women in screening and barriers. Women who were irregular or never participated, had barriers such as: lack of time (F = 9.51; p = 0,000), difficult access to Healthcare facilities (F = 11.29; p = 0.000), lack of knowledge about the Pap test procedure (F = 21.27, p = 0.000), discomfort (F = 9.36; p = 0.000), and anxiety of Pap test results (F = 3.35; p = 0.036). Women who regularly participated did not have prejudice when choosing a gynecologist, unlike the other two groups that preferred a female gynecologist (F = 3.61; p = 0.028). Conclusion: This study showed that the level of knowledge about CC and Pap test in women is an important factor associated with regular participation in screening. It is necessary to take educational measures in order to raise awareness of women regarding risk factors, as well as to overcome fear and shame, with the ultimate aim to reduce frequency and mortality rate caused by CC in Serbia.