Evolutional changes in homeotic gene functions have contributed to segmental diversification of arthropodan limbs, but crucial molecular changes have not been identified to date. The first leg of the crustacean Daphnia lacks a prominent ventral branch found in the second to fourth legs. We show here that this phenotype correlates with the loss of Distal-less and concomitant expression of Antennapedia in the limb primordium. Unlike its Drosophila counterpart, Daphnia Antennapedia represses Distal-less in Drosophila assays, and the protein region conferring this activity was mapped to the N terminal region of the protein. The results imply that Dapnia Antennapedia specifies leg morphology by repressing Distal-less, and this activity was acquired through a change in protein structure after separation of crustaceans and insects.