Female mate rejection acts as a major selective force within species, and can serve as a reproductive barrier between species. In spite of its critical role in fitness and reproduction, surprisingly little is known about the genetic or neural basis of variation in female mate choice. Here, we identify
fruitless
as a gene affecting female receptivity within
Drosophila melanogaster
, as well as female
Drosophila simulans
rejection of male
D. melanogaster
. Of the multiple transcripts this gene produces, by far the most widely studied is the sex-specifically spliced transcript involved in the sex determination pathway. However, we find that female rejection behaviour is affected by a non-sex-specifically spliced
fruitless
transcript. This is the first implication of
fruitless
in female behaviour, and the first behavioural role identified for a
fruitless
non-sex-specifically spliced transcript. We found that this locus does not influence preferences via a single sensory modality, examining courtship song, antennal pheromone perception, or perception of substrate vibrations, and we conclude that
fruitless
influences mate choice via the integration of multiple signals or through another sensory modality.