We have previously demonstrated a function for Neuroglian and Semaphorin1a in Drosophila giant fiber circuit formation. Both molecules are required for guiding the giant fibers out of the brain and have distinct functions during giant synapse formation. In this study we characterized the effects of various combinations of Neuroglian and Semaphorin1a gain and loss of function backgrounds on giant fiber circuitry formation. We found that Neuroglian and Semaphorin1a genetically interact with each other during axon guidance as well as during synapse formation. Our experiments revealed that during pathfinding of the giant fibers out of the brain, Neuroglian function seems to be dependent on Semaphorin1a. In contrast, during giant fiber synapse formation we observed that Semaphorin1a signaling as a receptor can be altered by Neuroglian in the same cell. In summary, our findings suggest that Neuroglian and Semaphorin1a can regulate each other's function in cis and that the resultant signaling output is possibly different during guidance and synapse formation.