BackgroundThe practice of exclusive breastfeeding (EBF) is influenced by maternal knowledge and attitudes as well as socio-demographic and cultural factors. This study assessed knowledge, attitudes and practice of EBF among rural lactating mothers with infants aged 0–6 months. Factors associated to the practice of EBF were also investigated.MethodsThis cross-sectional study was conducted among 190 rural lactating mothers with infants aged 0–6 months seeking postnatal care at a health centre in Ghana. All data was collected using a questionnaire that contained both closed and open ended questions.ResultsAbout 26 % (n = 50) of the mothers were unable to correctly define EBF. The majority (92.6 %, n = 176) of the mothers said they felt good to EBF for 6 months, to breastfed on demand (99.5 %, n = 189) and did not have difficulties EBF (90 %, n = 171). Despite the generally positive attitude towards EBF, 42 % (n = 79) of the mothers did not EBF their babies. These mothers did not practice EBF because they misunderstood certain signs of the child to mean wanting to eat food or drink water, regarded breastmilk to be inadequate to meet the nutritional needs of the child and misunderstood healthcare professionals’ EBF advice. Higher maternal education was associated with higher likelihood of EBF (OR 3.5; 95 % CI 1.6, 7.7; p = 0.002). Mothers whose babies were younger than 3 months were more likely to EBF (OR 12.0; 95 % CI 4.4, 32.5; p < 0.001) than those having babies aged ≥ 3 months. Furthermore, higher knowledge of EBF was associated with the likelihood of EBF (OR 5.9; 95 % CI 2.6, 13.3; p < 0.001).ConclusionMothers’ knowledge and attitudes towards EBF were favourable but practice of EBF was suboptimal. This study adds additional evidence that knowledge of EBF, child’s age and maternal level of education are important determinants of the practice of EBF. Beyond dissemination of health messages, healthcare professionals should pay more counselling attention to less educated mothers, and also older children’s caregivers.