The Masculinizer gene (Masc) encodes a CCCH tandem zinc finger protein essential for masculinization and dosage compensation in the silkworm Bombyx mori. Previously we identified a Masc orthologue from the crambid Ostrinia furnacalis (OfMasc) and observed its masculinizing activity in the B. mori cultured cell line BmN-4. However, the role of OfMasc in masculinization of O. furnacalis has not been assessed. In this study, we unexpectedly discovered that all of the male larvae that escaped from Wolbachia-induced embryonic male-killing by OfMasc cRNA injection expressed the female-type splicing variants of O. furnacalis doublesex (Ofdsx). To clarify the role of OfMasc in the masculinization process in vivo, we established a system to monitor both sex chromosome- and dsx splicing-based sexes from a single O. furnacalis embryo. Using this system, we investigated the effects of OfMasc knockdown in early embryos on Ofdsx splicing and found that depletion of OfMasc mRNA in male embryos induced the production of the female-type splicing variants of Ofdsx. This result indicates that OfMasc is required for masculinization in O. furnacalis, and that the Masc protein possesses masculinizing activity in an insect species that is phylogenetically distant from Bombycidae. (C) 2018 Elsevier Inc. All rights reserved.