<p>Breast cancer is the most common type of cancer among women, particularly in low and middle-income countries. Breast self-examination is one of the non-invasive methods of breast cancer screening in which a woman looks at her breast for any abnormal findings. Knowledge and practice of breast self-examination increase early diagnosis of breast cancer, which raises the chances for successful treatment and reduction of breast cancer mortality in Ethiopia. Therefore, the objective of this study was to assess knowledge and practice of breast self-examination among female undergraduate students in Jigjiga City. An institution-based cross-sectional study was conducted among 407 randomly selected female students. Data were collected using a structured and pretested questionnaire and entered into SPSS Version 24 for analysis. Descriptive, and bivariate analysis was conducted and variables having <i>p</i> &lt; 0.02 was selected for multivariable logistic regression analyses. The strength of statistical association between breast self-examination and independent variables was measured by an adjusted odds ratio and 95% confidence intervals, and statistical significance was declared at p-value &lt; 0.05. The study revealed that about 28.9% of participants had good knowledge of breast self-examination, while only 17.7% practiced it. Better knowledge was linked to younger age (<i>p</i> = 0.03), discussions about breast self-examination (<i>p</i> = 0.001), knowing someone with breast problems (<i>p</i> = 0.02), and receiving information from colleagues (<i>p</i> = 0.012). Practice was more common among students in higher academic years (<i>p</i> = 0.04), those with a family history of breast cancer (<i>p</i> = 0.011), and those who received information from colleagues (<i>p</i> = 0.001), and health professionals (<i>p</i> = 0.01). The level of knowledge and practice of breast self-examination by undergraduate female students was very low. Age, discussion about breast self-examination, knowing someone diagnosed with breast problems, colleagues as sources of information, academic year, and family history of breast cancer appeared to be significantly influencing the knowledge and practice of breast self-examination. Implementations are needed in addressing young females, making awareness and advocacy campaigns about breast self-examination through different media by stakeholders to increase early diagnosis of breast cancer, which raises the chances for successful treatment in Ethiopia.</p>