Ebola virus is a member of Filoviridae family of viruses that causes fetal hemorrhagic fever in human. Matrix protein VP40 of the Ebola virus is involved in multiple stages of viral maturation processes. In order to fully understand the interacting partners of VP40 in host cells, we applied proximity-dependent biotin-identification (BioID) approach to systematically screen for potential proteins at different time points of VP40 expression. By immunoprecipitation and subsequent proteomics analysis, we found over 100 candidate proteins with various cellular components and molecular functions. Among them, we identified Rab14 GTPase that appears to function at the late stage of VP40 expression. Imaging studies demonstrated that VP40 and Rab14 have substantial colocalization when expressed in HeLa cells. Overexpression of the dominant-negative Rab14(S25N) diminished the plasma membrane (PM) localization of VP40. In addition, we found that secreted VP40 protein can be endocytosed into Rab14 positive compartments. In summary, our study provides evidence that Rab14 is a novel regulator of the intracellular trafficking of Ebola virus matrix protein VP40 in HeLa cells. (C) 2020 Elsevier Inc. All rights reserved.