A breast screening program may help to reduce cancer mortality rates among women. However, the use of the screening program by women in Madinah city is low, and studies examining its practice and barriers of low uptake are sparse. To identify breast cancer knowledge, practice and screening barriers among women attending primary health centers (PHC) in Madinah, Saudi Arabia. A primary health center-based cross-sectional study was performed in Madinah city in 2015. A multistage stratified cluster sample was obtained and included 465 women (15 years and older) from five PHC. Data concerning socio-demographics, knowledge about breast cancer, and practice and barriers of mammography use were collected using a structured questionnaire. The mean age of the studied 465 women was 34.9 ± 12.2 years. Of these women, 27.7 and 38.5 % received mammography and performed breast self-examination, respectively. A high level of poor knowledge about breast cancer was detected in the overall studied women and those who never received a mammography, particularly knowledge related to the risk factors of breast cancer. The most important predictors of the barriers to mammography were incorrect beliefs about mammography and its procedures. A belief that mammography is painful was significantly associated with a 56 % reduction in its use (OR = 0.44; 95 % CI = 0.22-0.88). The high levels of poor knowledge about cancer breast observed in this study reflect the need for greater efforts to increase breast awareness education.