Exosomes are small extracellular vesicles of endocytic origin and are secreted into the extracellular space by fusion between intraluminal vesicle (ILV)-containing multivesicular bodies (MVBs) and the plasma membrane. It has recently been shown that a single cell such as an epithelial cell secretes distinct types of exosomes having different protein compositions (referred to as “exosomal heterogeneity”). Polarized epithelial cells have two distinct plasma membrane domains (an apical plasma membrane and a basolateral plasma membrane), which are separated by tight junctions. Two distinct exosome subtypes, i.e., apical exosomes and basolateral exosomes, are asymmetrically secreted from the apical side and basolateral side, respectively, of the cell. These two subtypes are independently regulated by distinct mechanisms in terms of ILV (exosome precursor) biogenesis and MVB transport to the plasma membrane. On the apical side, ILV formation is regulated by the ALIX–syntenin-1–syndecan-1 complex and the MVB formed is transported to the apical plasma membrane by small GTPases Rab27 and Rab37. On the basolateral side, ILV formation is regulated by the neutral sphingomyelinase 2 (nSMase2)-mediated ceramide production machinery, and the MVB formed is transported to the basolateral plasma membrane by Rab39–UACA–BORC. These mechanisms are also retained in nonpolarized cells and are likely to contribute to the genesis of exosomal heterogeneity in nonpolarized cells.