The urbanization of the city of Edo, the capital of premodern Japan, has been assumed to be not as a result of natural increase but that of in-migration although this assumption has never been verified. To obtain information on natural fertility in Edo, we analyzed stable carbon and nitrogen isotopes in 46 adult and 84 subadult human skeletons excavated from the Hitotsubashi site (1657-1683 AD: the early Edo period), Tokyo, Japan and reconstructed their breastfeeding period, one of the most important determinants of fertility. Adult females are significantly more depleted in (15) N by 0. 7‰ than adult males, suggesting a dietary differentiation between sexes and/or the effect of pregnancy. The changes in the nitrogen isotope ratios of subadults suggest that supplementary foods were introduced around the age of 0.2 years and weaning ended around 3.1 years, which agrees with descriptions in various historical documents of the period. The duration of breastfeeding in the Hitotsubashi population was relatively longer than those in modern industrial and traditional societies and four previously reported populations in medieval and in the industrial England. As later weaning closely associates with longer inter-birth interval for mothers, our data suggest a lower natural fertility for the Hitotsubashi population. Assuming that the proportion of married people was also lower in the major cities of the earlier Edo period, our results support the assumption that Edo developed and increased its population by attracting immigrants during urbanization.