Plant cuticle is the outermost layer of a plant that interacts with environment and protects plants from water loss and various biotic and abiotic stresses. However, its related functional mechanisms are not well studied. Our study first shows that one member of adenosine diphosphate (ADP) ribosylation factor (ARF) guanine nucleotide exchange factor (GEF) protein family of Arabidopsis plants is critical for cuticle formation and related defense against diverse pathogens, including the bacterium Pseudomonas syringae and fungus Botrytis cinerea. This protein is localized in the trans-Golgi network/early endosome (TGN/EE), where it regulates the endocytic cycling of plasma membrane localized proteins. Through Transmission Electron Microscopy (TEM), the mutant leaves showed thinner cuticular layer compared with wild-type plants and the cuticle ledge of stomata is almost missing. Through Scan Electron Microscopy (SEM), the mutant leaves showed more stomata with defective structures. The GC-MS analysis shows that the fatty acid and cutin contents are significantly reduced in mutant plants. Thus, we discovered that the vesicle trafficking related component might be involved in the transport of cuticle related compounds/proteins for the cuticle formation and related defense function. Our further study on the detailed mechanisms and related networks may facilitate the development of efficient strategies to enhance plant health and yield through genetic manipulation of the plant cuticle, thus reducing the agricultural chemical applications to benefit the environment and human health.