The Drosophila fruitless (fru) gene is regarded as a master regulator of the formation of male courtship circuitry, yet little is known about its molecular basis of action. We show that roundabout 1 (robo1) knockdown in females promotes formation of the male-specific neurite in sexually dimorphic mAL interneurons and that overexpression of the male-specific Fru(BM) diminishes the expression of Robo1 in the fly brain. Our electrophoretic mobility shift and reporter assays identify the 42-bp segment encompassing the palindrome sequence T T C G C T G C G C C G T G A A in the 5' UTR of robo1 exon1 as the Fru(BM)-responsive element. We find that ∼10-bp deletions in the palindrome sequence induce a loss of the male-specific neurite and disrupt male courtship patterns. This study paves the way for a thorough understanding of the mechanism whereby Fru proteins orchestrate transcription for the formation of courtship circuitry.