Autophagy is required for neurulation, and autophagy activators with minimal toxicity, such as the natural compound trehalose, a nonreducing disaccharide, possess high therapeutic value. To determine whether trehalose directly induces autophagy, FITC-labeled trehalose was used for tracing its presence in autophagosome complexes. Trehalose was as potent as rapamycin and starvation in inducing de novo autophagosome formation and increasing autophagosome flux in GFP-LC3 reporter cells and C17.2 neural stem cells. Trehalose effectively reversed high glucose-suppressed autophagy and reduced p62 protein expression. Trehalose abolished the disruption of autophagosome complexes under high glucose conditions in vitro and maternal diabetes in vivo. Autophagosomes induced by trehalose were functionally active, forming mitophagy and reticulophagy in removing damaged cellular organelles in neuroepithelial cells exposed to maternal diabetes. Thus, trehalose directly participated in functional autophagosome generation by incorporating itself into autophagosomes. These findings provide the mechanistic basis for the use of trehalose in preventing disruptive autophagy-associated pathogenesis.