Cre-mediated recombination is widely used to manipulate defined genes spatiotemporally in vivo. The present study evaluated the Cre/loxP system in Bombyx mori by establishing two transgenic lines. One line contained a Cre recombinase gene controlled by a sericin-1 gene (Ser1) promoter. The other line contained a loxP-Stop-loxP-DsRed cassette driven by the same Ser1 promoter. The precise deletion of the Stop fragment was found to be triggered by Cre-mediated site-specific excision, and led to the expression of DsRed fluorescence protein in the middle silk glands of all double-transgenic hybrids. This result was also confirmed by phenotypical analysis. Hence, the current study demonstrated the feasibility of Cre-mediated site-specific recombination in B. mori, and opened a new window for further refining genetic tools in silkworms.