Up-regulation of Centrosomal Protein 55 (CEP55) in cancer cells increases malignancy, and the protein can be transferred via exosomes. However, the mechanism of how CEP55 is delivered to exosomes is unknown. In this study, we addressed this issue and analysed trafficking of EGFP-CEP55 from early to late endosomes by using high-resolution microscopy. Our data show that endogenous as well as EGFP-CEP55 appeared as dot-like structures in cancer cells. However, we did not find an internalization of CEP55 into early Rab5- and late Rab7-positive endosomes but only into secretory late CD63-positive endosomes. In addition, an association of the CEP55 dots with the endoplasmic reticulum and with ALG-2-interacting protein X (Alix) dots was detected. Moreover, mutation of the CEP55-Alix interaction site strongly reduced the formation of CEP55 dots as well as CEP55 localization in extracellular vesicles. In summary, our data indicate that delivery of CEP55 into exosomes does not occur by the canonical early-to-late endosome pathway but by Alix-mediated recruitment to secretory late secretory CD63 endosomes.