Hatching behavior is a key target in silkworm (Bombyx mori) rearing, especially for the control of Lepidoptera pests. According to previous research, hatching rhythms appear to be controlled by a clock mechanism that restricts or “gates” hatching to a particular time. However, the underlying mechanism remains elusive. Under 12‐h light:12‐h dark photoperiod (LD) conditions, the transcriptional levels of the chitinase5 (Cht5) and hatching enzyme‐like (Hel) genes, as well as the enzymatic activities of their gene products, oscillated in time with ambient light cycles, as did the transcriptional levels of the cryptochrome 1, cryptochrome 2, period (per), and timeless genes, which are key components of the negative feedback loop of the circadian rhythm. These changes were related to the expression profile of the ecdysteroid receptor gene and the hatching behavior of B. mori eggs. However, under continuous light or dark conditions, the hatching behavior, the expression levels of Cht5 and Hel, as well as the enzymatic activities of their gene products, were not synchronized unlike under LD conditions. In addition, immunohistochemistry experiments showed that light promoted the translocation of PER from the cytoplasm to the nucleus. In conclusion, LD cycles regulate the hatching rhythm of B. mori via negative feedback loop of the circadian oscillator.