The prothoracicostatic peptide isolated from the larval brains of the silkworm, Bombyx mori (Bom-PTSP) is a novel insect neuropeptide, whose primary structure is AWQDLNSAW-NH2. It inhibits ecdysteroid synthesis in the prothoracic glands (PGs) of silkworms both in vitro and in vivo. The percentages of the secondary structures, α-helix, β-sheet, β-turn and random coil, of PTSP and its analog W-1475, SWQDLNSVW-NH2 (belongs to the W(X6)W-amides family) were analyzed by circular dichroism (CD) and nuclear magnetic resonance (NMR). The structural differences resulted in the differences of biological activities on ecdysteroid biosynthesis in PGs. The biological activities of Bom-PTSP and its analog peptides on ecdysteroid biosynthesis in PGs by in vitro bioassay were examined at different concentration and the maximum inhibition rate of W-1475 was only half of that of PTSP. The results from in vivo bioassay also showed changes at the amino acid sequence of Bom-PTSP resulted in the change of the secondary structure, and accordingly, decrease or loss of its biological activity on ecdysteroidonenesis. These results help to establish the base to elucidate and demonstrate farther the relationship between chemic spatial structures and functions of PTSP and analog peptides of PTSP.