To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca²⁺ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca²⁺. The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca²⁺ binding assay indicated that rCNB shows affinity for Ca²⁺. The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca²⁺ or efflux Ca²⁺ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.