Extracellular vesicles (EVs) play crucial roles in cell-cell communication, but the biogenesis of large EVs has remained elusive. Here, we show that the biogenesis of large EVs (>800nm-2um) occurs predominantly through the completion of successful cytokinesis, and the majority of large extracellular vesicles are midbody remnants (MBRs) with translation activity, and the unique marker MKLP1. Blocking the cell cycle or cytokinesis, genetically or chemically, significantly decreases MBRs and large (800nm-2um), medium (500-800nm), and small (<300nm) EVs, suggesting that proliferative cells can also generate all sizes of EVs. The canonical EV markers including CD9, CD63, CD81 localize to the spindle midzone, midbody and MBRs, suggesting that these markers are not specific for detecting EVs exclusively. Importantly, all commonly used EV isolation methods isolate MBRs, confounding previous EV research. Lastly, isolated MBRs maintain translation activity regardless of the isolation method. We propose a model for the biogenesis of extracellular vesicles throughout the cell cycle and suggest that some large EVs are primarily generated from mitotic cells. The discovery of MBRs as a unique class of large, translating EVs has implications for using them as cancer diagnostic markers and for engineering them for therapeutic cargo delivery during mitosis.