Diploid sexual reproduction involves segregation of allelic pairs, ensuring equal representation of genotypes in the gamete pool. Some genes, however, are able to "cheat" the system by promoting their own transmission. The Segregation distorter (Sd) locus in Drosophila melanogaster males is one of the best-studied examples of this type of phenomenon. In this system the presence of Sd on one copy of chromosome 2 results in dysfunction of the non-Sd-bearing (Sd(+)) sperm and almost exclusive transmission of Sd to the next generation. The mechanism by which Sd wreaks such selective havoc has remained elusive. However, its effect requires a target locus on chromosome 2 known as Responder (Rsp). The Rsp locus comprises repeated copies of a satellite DNA sequence and Rsp copy number correlates with sensitivity to Sd. Under distorting conditions during spermatogenesis, nuclei with chromosomes containing greater than several hundred Rsp repeats fail to condense chromatin and are eliminated. Recently, Rsp sequences were found as small RNAs in association with Argonaute family proteins Aubergine (Aub) and Argonaute3 (AGO3). These proteins are involved in a germline-specific RNAi mechanism known as the Piwi-interacting RNA (piRNA) pathway, which specifically suppresses transposon activation in the germline. Here, we evaluate the role of piRNAs in segregation distortion by testing the effects of mutations to piRNA pathway components on distortion. Further, we specifically targeted mutations to the aub locus of a Segregation Distorter (SD) chromosome, using ends-out homologous recombination. The data herein demonstrate that mutations to piRNA pathway components act as enhancers of SD.