Selenophosphate synthetase (Sps), the product of the SelD gene, produces a biologically active selenium donor compound from ATP and selenide. We have isolated and characterized the Sps gene from Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae), an economically important insect. The resulting 1601 bp cDNA sequence contains an open reading frame of 1209 bp encoding a polypeptide of 402 amino acids, with 87% sequence identity to that from Drosophila melanogaster (Meigen). Semiquantitative reverse transcription-polymerase chain reaction (PCR) analysis showed that the Sps gene was transcribed during four developmental stages (egg, larva, pupa, and adult) and in all the tissues tested (blood, fat body, midgut, silk glands, body wall, spermaries and ovaries), suggesting that ApSps plays an important role in the development of A. pernyi. From a database search, Sps protein homologs were found in prokaryotes and eukaryotes, including bacteria, fungi, invertebrates and vertebrates, with 47–98% amino acid sequence identities between eukaryotes, suggesting that they were highly conserved during the evolution of eukaryotes. Phylogenetic analysis, based on Sps protein homolog sequences, clearly separated the known bacterial, fungal, invertebrate and vertebrate Sps proteins, consistent with the topology tree of classical systematics, suggesting the potential value of the Sps protein sequence in phylogenetic inference.