A longer breastfeeding duration provides various positive effects in subadult health because of abundant immunological factors and nutrients in human breast milk, and decreases the natural fertility of a population through lactational amenorrhea. In this study, we measured stable carbon and nitrogen isotope ratios in the bone collagen of three adults and 45 subadults from the Yuigahama-minami site (from 12th to 14th century) in Kamakura, the early medieval capital of Japan. Marine foods, C3 -based terrestrial foods, and freshwater fish are the primarily protein sources for adults. The changes in the nitrogen isotope ratios of subadults suggest that the relative dietary protein contribution from breast milk started to decrease from 1.1 years of age and ended at 3.8 years. The age at the end of weaning in the Yuigahama-minami population was greater than that in the typical non-industrial populations, a premodern population in the Edo period Japan, and medieval populations in the UK. Skeletons of townspeople from medieval Kamakura indicate severe nutritional stress (e.g., enamel hypoplasia and cribra orbitalia), yet this longer duration of breastfeeding did not compensate adverse effects for nutritional deficiency. The longer breastfeeding period may have been a consequence of complementary food shortage and bad health of subadults. Kamakura experienced urbanization and population increase in the early medieval period. The younger age-at-death distribution and high nutritional stresses in the Yuigahama-minami population and later weaning, which is closely associated with longer inter-birth interval for mothers, suggests that Kamakura developed and increased its population by immigration during urbanization.