Golden goal (Gogo) is a cell surface protein that is crucial for proper synaptic layer targeting of photoreceptors (R cells) in the Drosophila visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporary and final layer targeting of R-cell axons through its cytoplasmic activity. However, it is not known how Gogo activity is regulated. Here, we show that a conserved Tyr-Tyr-Asp (YYD) tripeptide motif in the Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to abolish Gogo function. We demonstrate that the YYD motif is a phosphorylation site and that mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in axon stopping at the temporary targeting layer, and dephosphorylation is crucial for final layer targeting in collaboration with Fmi. Therefore, both temporary and final layer targeting strongly depend on the Gogo phosphorylation status. Drosophila Insulin-like receptor (DInR) has been reported to regulate the wiring of photoreceptors. We show that insulin signaling is a positive regulator, directly or indirectly, of YYD motif phosphorylation. Our findings indicate a novel mechanism for the regulation of Gogo activity by insulin signaling-mediated phosphorylation. We propose the model that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that controls developmental timing in axon targeting.