Aim: This study aimed to compare the factors influencing exclusive breastfeeding (EBF) practices between rural and urban areas in Anhui Province, China, with a focus on identifying biophysical, social, and cultural determinants. Background: The World Health Organization and UNICEF both recognize exclusive breastfeeding as the optimal method of infant nutrition. In China, breastfeeding practices vary significantly between urban and rural areas due to factors such as cultural beliefs, healthcare services, socioeconomic status, and employment. This study aims to fill the research gap in Anhui Province, a rapidly urbanizing region. Methods: A quantitative comparative research design was employed, with data collected from 200 lactating mothers (100 from urban and 100 from rural areas) aged 18 years and above. The study used self-administered questionnaires that assessed demographic characteristics, biophysical, social, and cultural factors influencing EBF practices. Poisson regression analysis was conducted to examine the association between urbanicity and breastfeeding practices, adjusting for key variables such as age, employment, and income. Results: The study indicated that mothers in both rural and urban Anhui Province engaged more in exclusive breastfeeding (EBF) when supported by biophysical and social factors. Cultural influences were notably greater for rural mothers. Biophysical and social factors showed no significant differences between the groups. Regression analysis identified maternal age (31–35 years) and female infant sex as significant predictors of EBF. Employment negatively affected social support in both environments, while education and income impacted cultural attitudes towards EBF differently. Conclusion: Regardless of location, mothers who exclusively breastfeed highly value biophysical, social, and cultural factors. In urban areas, factors such as age, employment, and higher income significantly influence breastfeeding, while in rural areas, infant gender, marital status, income, and employment status are more crucial. Furthermore, both urban and rural mothers consider biophysical and social factors, but cultural factors are more prominent in the breastfeeding practices of rural mothers than urban mothers.