The biogenic monoamine 5-hydroxytryptamine (5-HT) is an ancient intracellular signaling molecule widely distributed in all animals with nervous systems, and has been implicated in principal behaviors. Tryptophan hydroxylase (TRH) induces a highly specific catalytic reaction that converts L-tryptophan (tryptophan) to 5-hydroxy-L-tryptophan (5-HTP) that is subsequently used as a substrate by aromatic L-amino acid decarboxylase (DDC) to form 5-HT. Five-HT is an ancient intracellular signaling molecule that is widely distributed in the animal kingdom and has been implicated in regulating the behaviors of animals with nervous systems. However, the role of TRH in Lepidoptera is not well understood. In this study, we cloned 1 667 bp cDNAs of Bombyx mori TRH (BmTRH), which contains a 1 632 bp open reading frame (ORF). Homology analysis revealed that BmTRH shared high amino acid identity with Homo sapiens TPH and Drosophila TRH (DmTRH). The high homology (70%) of BmTRH with DmTRH suggested that BmTRH could have a function similar to DmTRH. Gene expression analysis revealed that BmTRH was mainly expressed in head and central nervous (CNS). Moreover, immunohistochemistry and Western blotting analyses showed that BmTRH was detected only in larval nervous tissues. Taken together, our results indicate that BmTRH could likely function in the regulation of neural activities in B. mori. The transcripts of B. mori decarboxylase (BmDDC) and B. mori phenylalanine hydroxylase (BmPAH) whose proteins had TRH activity, were also expressed in the CNS tissues, indicating that unlike in Drosophila, two distinct mechanisms likely regulate 5-HT synthesis in silkworm.