Early diagnosis of breast cancer is the best approach towards its control that may result in alleviating related mortality and morbidity. This study aimed to evaluate knowledge about breast cancer and both practices and perceived barriers to breast self-examination among female university students in Bangladesh. A cross-sectional study was carried out with 400 female students of Jahangirnagar University, Bangladesh. Participants were sampled from female dormitories at the university from January to April 2020. Proportionate stratified random sampling was conducted to calculate the study sample from each dormitory. A validated semi-structured self-reported questionnaire was employed to collect data from participants during the survey periods. The questionnaire consisted of demographic variables, items about knowledge about breast cancer, breast self-examination practices and its barriers. We applied descriptive and inferential statistics and data were analyzed using the Statistical Package for the Social Sciences (SPSS). Participants were aged between 18-26 years and comprised university students of first year (20%), second year (24%), third year (22%), fourth year (21%) and Master's (14%). 18% of them reported positive family history (mother, aunt, sister/cousin, grandmother) of breast cancer. The overall mean score of total knowledge items was 15 (SD = 3) out of 43, with an overall correct rate of 34%. The mean score of total knowledge items was significantly higher (p<0.001) among Master's students and students with family members who have had breast cancer. Only one in five students (21%) ever practiced breast self-examination. The mean score of practice of breast self-examination was significantly higher (p<0.001) among participants who reported having family member of breast cancer. Total knowledge score about breast cancer and practice of breast self-examination were significantly correlated with each other (r = 0.54; p<0.001). About 33% participants reported 'lack of knowledge' as the main barrier to practicing breast self-examination followed by 'I do not have the symptoms' (22%), and 'shyness/uncomfortable feelings' (17%). The study revealed low levels of knowledge about breast cancer and low breast self-examination practices. Our findings highlight the need to develop, implement and promote socially, culturally and demographically appropriate educational interventions programs aimed at breast cancer and breast self-examination awareness and practice in Bangladesh.