ROBO3 antagonizes ROBO1/ROBO2 function to prevent their response to SLIT, thus allowing cells that are expressing ROBO1/ROBO2 to progress towards and across the floor plate. Exactly how ROBO3 interferes with ROBO1/ROBO2 function is not yet clear (Chen et al. 2008). It was shown that ROBO3 isoform ROBO3.1 reduces the amount of ROBO1 and ROBO2 at the cell surface and suggested that ROBO3.1 acts by directing ROBO1 and ROBO2 to late endosome- and lysosome-dependent degradation pathway (Li et al. 2014). Direct binding of ROBO3.1 to ROBO2 was demonstrated (Li et al. 2014).

During commissural axon midline crossing in Drosophila, Robo1 signaling can also be antagonized with Robo2 expressed in trans. Extracellular domains of Robo1 and Robo2 may interact through their Ig domains, preventing Robo1 activation by Slits and interfering with axon repulsion (Evans et al. 2015).