The amount of genetic variation for fitness within populations tends to exceed that expected under mutation-selection-drift balance. Several mechanisms have been proposed to actively maintain polymorphism and account for this discrepancy, including antagonistic pleiotropy (AP), where allelic variants have opposing effects on different components of fitness. Here we identify a non-coding indel polymorphism in the fruitless gene of Drosophila melanogaster and measure survival and reproductive components of fitness in males and females of replicate lines carrying each respective allele. Expressing the fruitless region in a hemizygous state reveals a pattern of AP, with one allele generating greater reproductive fitness and the other conferring greater survival to adulthood. Different fitness effects were observed in an alternative genetic background, which may reflect dominance reversal and/or epistasis. Our findings link sequence-level variation at a single locus with complex effects on a range of fitness components, thus helping to explain the maintenance of genetic variation for fitness. Transcription factors, such as fruitless, may be prime candidates for targets of balancing selection since they interact with multiple target loci and their associated phenotypic effects.