It is unknown whether maternal health and nutrition are related to human milk composition or growth and development of infants and children. Here, we describe a protocol for a prospective five-year cohort study to clarify (i) how maternal health and nutrition, socioeconomic factors, and lifestyles affect human milk composition, and (ii) whether these are associated with growth and development of infants and children. In our study, we recruited 1210 Japanese mothers with singleton pregnancies from 73 obstetrics clinics and hospitals across Japan, between 2014 and 2019. We will measure the following: health information regarding maternal-child dyads using a self-administered questionnaire, maternal nutrition during breastfeeding using a Brief self-administrated Diet History Questionnaire, the development of infants and children using the Kinder Infant Development Scale, and the stress related to child rearing using the Mother’s Child Care Stress Scale. Simultaneously, we will collect human milk every 2 months during the first year after birth to measure its composition and levels of macronutrients. This study will generate useful data to investigate whether health status, nutritional status, lifestyle, and socioeconomic factors affect human milk composition and the growth and development of infants and children.