Hetero- and homodimerization of 14-3-3 proteins demonstrate distinctive functions in mammals and plants. Trypanosoma brucei 14-3-3I and II (Tb14-3-3I and II) play pivotal roles in motility, cytokinesis and the cell cycle; however, the significance and the mechanism of Tb14-3-3 dimerization are remained to be elucidated. We found that ectopically expressed epitope-tagged Tb14-3-3I and II proteins formed hetero- and homodimers with endogenous Tb14-3-3I and II proteins. However, we also found the ability to form hetero- or homodimers between Tb14-3-3I and II proteins was clearly affected by the sequence and location of the epitope tag used. We found a blue native polyacrylamide gel electrophoresis system followed by western blotting may distinguish monomer from dimer structure, and stable from unstable conformation of Tb14-3-3. Combined with co-immunoprecipitation results, we revealed that Tb14-3-3 proteins mainly existed as heterodimeric form. Furthermore, co-overexpression of Tb14-3-3I and II proteins in T. brucei induced aberrant numbers of organelles in cells, but overexpression of either isoform alone rarely produced such morphology. These results suggest that heterodimers play more significant roles than homodimers not only in the maintenance of steady-state levels of the 14-3-3 proteins but also in the regulation of cytokinesis.