SignificanceTri-longin domain (TLD) RabGEFs represent a group of protein complexes that play crucial roles in the organization of endosomes, lysosomes, and lysosome-related organelles. During evolution, the TLD family has diversified from one dimeric complex in yeast to three complexes with up to four subunits in metazoans. How this expansion contributes to different functions and regulation remains elusive. Our cryo-electron microscopy (cryo-EM) structure of the trimeric Mon1–Ccz1–Bulli TLD RabGEF from fruit flies provides a framework to address these questions and to understand the implications for the organization of the endolysosomal system.