Drosophila melanogaster adult males perform an elaborate courtship ritual to entice females to mate. fruitless (fru), a gene that is one of the key regulators of male courtship behavior, encodes multiple male-specific isoforms (FruM). These isoforms vary in their carboxy-terminal zinc finger domains, which are predicted to facilitate DNA binding. By over-expressing individual FruM isoforms in fru-expressing neurons in either males or females and assaying the global transcriptional response by RNA-sequencing, we show that three FruM isoforms have different regulatory activities that depend on the sex of the fly. We identified several sets of genes regulated downstream of FruM isoforms. Overall design: RNA seqeuncing was performed on mRNA derived from adult male or female heads, for a total of 39 samples. These samples included two wild type genotypes (Berlin and Canton-S), two transheterozygous mutants for fru P1 (Df(3R)P14/Df(3R)fru4-40 and fruw12/ Df(3R)ChaM5), and 3 overexpressing genotypes (fru P1-Gal4: UAS-FruMA, UAS-FruMB, UAS-FruMC). There were at least 3 replicates from biological samples for all sex by genotype combinations.