Production of vitamin C has been traditionally based on the Reichstein process and the two-step process. However, the two processes share a common disadvantage: vitamin C cannot be directly synthesized from D-glucose. Therefore, significant effort has been made to develop a one-step vitamin C fermentation process. While, 2-KLG, not vitamin C, is synthesized from nearly all current one-step fermentation processes. Vitamin C is naturally synthesized from glucose in Arabidopsis thaliana via a ten-step reaction pathway that is encoded by ten genes. The main objective of this study was to directly produce vitamin C from D-glucose in Escherichia coli by expression of the genes from the A. thaliana vitamin C biosynthetic pathway. Therefore, the ten genes of whole vitamin C synthesis pathway of A. thaliana were chemically synthesized, and an engineered strain harboring these genes was constructed in this study. The direct production of vitamin C from D-glucose based on one-step fermentation was achieved using this engineered strain and at least 1.53 mg/L vitamin C was produced in shaking flasks. The study demonstrates the feasibility of one-step fermentation for the production of vitamin C from D-glucose. Importantly, the one-step process has significant advantages compared with the currently used fermentation process: it can save multiple physical and chemical steps needed to convert D-glucose to D-sorbitol; it also does not involve the associated down-streaming steps required to convert 2-KLG into vitamin C.