To clarify the control mechanism of the catalytic activity of casein kinase 2 (CK2) during early embryonic development in the silkworm, Bombyx mori, we attempted an in-vitro functional analysis by using the recombinant α and β subunits of B. mori CK2 (rBmCK2α and rBmCK2β) produced in a bacterial system. The renatured rBmCK2α possessed protein kinase activity. When rBmCK2α and rBmCK2β were reconstituted in an approximate 1:1 molar ratio, the catalytic activity was almost the same as that of rBmCK2α alone. The catalytic activity of rBmCK2α was inhibited by polylysine, which is one of the activators of CK2 activity. However, when using the reconstituted rBmCK2α and rBmCK2β (rBmCK2), activation by polylysine was observed. We examined the influence of sorbitol and 3-hydroxykynurenine (3-OHK), which are contained mainly in diapause eggs, on the phosphorylation activity of rBmCK2. Three-OHK inhibited rBmCK2 activity, but sorbitol had no effect on it. Furthermore, a functional analysis using rBmCK2α and β subunits of Drosophila melanogaster CK2 revealed that a difference in the C-terminal amino acid of the CK2β subunit influenced the phosphorylation activity of rBmCK2α. These results may provide new insights for clarifying the control mechanism of B. mori casein kinase 2 in eggs.