The initial signal that governs sex determination is highly variable among insects. A homolog of Nix, the male-determining factor in Aedes aegypti, was previously found in the Asian tiger mosquito Ae. albopictus. Here we show that the Ae. albopictus Nix (AalNix) is more complex in gene structure and splice isoforms than its Ae. aegypti homolog (AaeNix). AalNix shows a similar transcription profile compared to AaeNix. CRISPR/Cas9mediated knockouts of AalNix in vivo and in the Ae. albopictus C6/36 cells lead to a shift of dsx and fru splicing towards the female isoforms. G(o) knockout males showed feminization and deformities including feminized antennae, absence or partial absence of gonocoxites, gonostyli, testes and accessory glands, and the formation of ovaries. Despite similar to 70 MY of divergence, Nix functions as a conserved male-determining factor in the two most important arboviral vectors, namely Ae. aegypti and Ae. albopictus.