The pigmentation of insects has served as an excellent model for the study of morphological trait evolution and developmental biology. The melanism (mln) mutant of the silkworm Bombyx mori is notable for its strong black coloration, phenotypic differences between larval and adult stages, and its widespread use in strain selection. Here, we report the genetic and molecular bases for the formation of the mln morphological trait. Fine mapping revealed that an arylalkylamine N-acetyltransferase (AANAT) gene co-segregates with the black coloration patterns. Coding sequence variations and expression profiles of AANAT are also associated with the melanic phenotypes. A 126 bp deletion in the mln genome causes two alternatively spliced transcripts with premature terminations. An enzymatic assay demonstrated the absolute loss of AANAT activity in the mutant proteins. We also performed RNA interference of AANAT in wild-type pupae and observed a significant proportion of adults with ectopic black coloration. These findings indicate that functional deletion of this AANAT gene accounts for the mln mutation in silkworm. AANAT is also involved in a parallel melanin synthesis pathway in which ebony plays a role, whereas no pigmentation defect has been reported in the Drosophila model or in other insects to date. To the best of our knowledge, the mln mutation is the first characterized mutant phenotype of insects with AANAT, and this result contributes to our understanding of dopamine metabolism and melanin pattern polymorphisms.