Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network-recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner.