The Japanese bumblebee Bombus cryptarum florilegus is threatened with extinction, and for the first time, a detailed study of their nests has been successfully conducted in the Nemuro Peninsula, Hokkaido Island. This bumblebee nested in a dead grass nest of small animals built in a closed space covered by grass on the ground. The number of cells in the nest was 511. It was estimated that 76 new queens were born in this nest, which was the largest among bumblebee species in Hokkaido. Adult new queens (average 5.36 mm in head width) were larger than those of workers (average 4.55 mm in head width). The sex ratio of the mature nest examined in this study was highly female-biased, suggesting the possibility of a split sex ratio in the Nemuro population. The nesting biology of B. cryptarum florilegus revealed in this study is essential for future conservation.