In Drosophila, RotundRacGAP/RacGAP(84C) is critical to retinal organisation and spermatogenesis. We show that eye-directed expression of RacGAP(84C) or its GTPase activating protein (GAP) domain induces a dominant rough eye phenotype which we used as a starting point in a gain-of-function screen to identify new partners of RacGAP(84C). Proteins known to function in Ras, Rho and Rac signalling were identified confirming the essential role of RacGAP(84C) in crosstalk between GTPases. Other potential RacGAP(84C) partners identified by the screen are implicated in signal transduction, DNA remodelling, cytoskeletal organisation, membrane trafficking and spermatogenesis. This latter class includes the serine/threonine kinase Center divider (Cdi), which is homologous to the human LIM kinase, Testis specific kinase 1 (TESK1), involved in cytoskeleton control through Cofilin phosphorylation. Eye-directed expression of cdi strongly suppressed the phenotypes induced by either RacGAP(84C) gain-of-function or by the dominant negative form of Rac1, Rac1N17. These results are consistent with Cdi being a specific downstream target of Rac1. We showed that Rac1 and cdi are both expressed in Drosophila testis and that homozygous Rac1 mutants exhibit poor fertility that is further reduced by introducing a cdi loss-of-function mutation in trans. Thus, results from a misexpression screen in the eye led us to a putative novel Rac1-Cdi-Cofilin pathway, regulated by RacGAP(84C), coordinating Drosophila spermatogenesis.