In this study we examined Roundabout signaling in the Drosophila embryonic hindgut.Slit and its receptors Roundabout (Robo) and Roundabout 2 (Robo2) localize to discrete regions in the hindgut epithelium and surrounding visceral mesoderm. Loss of robo, robo2 or slit did not disrupt overall hindgut patterning. However, slit and robo mutants showed a decrease in microvillus length on the boundary cells of the hindgut epithelium. Rescue and overexpression analysis revealed that robo is specifically required in the visceral mesoderm for correct microvillus length in the underlying hindgut epithelium. Expression of robo in the visceral mesoderm of robo mutant embryos restored normal microvillus length, while overexpression of robo resulted in an increase in microvillus length. Microvillus length was also increased in robo2 mutants suggesting that robo2 may antagonize robo function in the hindgut.Together, these results establish a novel, dose-dependent role for Robo in regulating microvilli growth and provide in vivo evidence for the role of the visceral mesoderm in controlling morphological changes in the underlying intestinal epithelium.