Background. Exclusive breastfeeding (EBF) is the gold standard of infant feeding practice which lasts up to 6 months postpartum. Not all infants are exclusively breastfed in developing countries, including Ethiopia. This study, therefore, assessed the magnitude and determining factors of EBF cessation practice among mothers at University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. Method. Institutional-based cross-sectional study design using a systematic random sampling technique was applied to select 344 mothers of infants aged 9 months came for measles vaccination. Pretested structured questionnaire was used to collect the data. Data were entered, cleaned, and analyzed by using SPSS version 21. Independent variables with a P value of <0.05 indicated association. Result. The magnitude of cessation of EBF was 21.5% with 95% CI (17.24-25.76). Maternal age ≤ 19 years [AOR=5.53; 95% CI (1.07-28.57)], civil servants [AOR=4.73; 95% CI (2.20-10.19)], illiterate husbands [AOR=3.76; 95% CI (1.13-12.49)], primi-para [AOR=2.42; 95% CI (1.22-4.79)], no postnatal follow up [AOR=2.62; 95% CI (1.44-4.80)], and having poor knowledge on breastfeeding benefits and composition of breastmilk [AOR=3.15; 95% CI (1.56-6.35)] were independent factors significantly associated with cessation of EBF. Conclusion and recommendation. The magnitude of cessation of EBF was high. Maternal age, parity, employment status, postnatal follow-up, and breastfeeding knowledge as well as spouse literacy level were independent factors significantly associated with cessation of EBF. Our study provides further impetus for empowering young and primi-para with breastfeeding knowledge, an extension of maternity leave time, and support for breastfeeding at the workplace.