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Background: Boehmeria nivea L. Gaud (Ramie) produces one of the longest natural fibers in nature. The bark of ramie mainly comprises of the phloem tissue of stem and is the raw material for fiber. Therefore, identifying the molecular regulation of phloem development is important for understanding of bast fiber biosynthesis and improvement of fiber quality in ramie., Results: In this study, we collected top bud (TB), bark from internode elongating region (ER) and bark from internode fully elongated region (FER) from the ramie variety Zhongzhu No. 1. Histological study indicated that these samples contain phloem tissues at different developmental and maturation stages, with a higher degree of maturation of phloem tissue in FER. RNA sequencing (RNA-seq) was performed and de novo transcriptome was assembled. Unigenes and differentially expressed genes (DEGs) in these three samples were identified. The analysis of DEGs by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed clear differences in gene expression between ER and FER. Some unigenes involved in secondary cell wall biosynthesis were up-regulated in both ER and FER, while unigenes for some cell wall components or cell wall modifications showed differential expression between ER and FER. In addition, the ethylene respond factors (ERFs) in the ethylene signaling pathway were up-regulated in FER, and ent-kaurenoic acid oxidase (KAO) and GA 20-oxidase (GA20ox) for gibberellins biosynthesis were up-regulated while GA 2-oxidase (GA2ox) for gibberellin inactivation was down-regulated in FER., Conclusions: Both morphological study and gene expression analysis supported a burst of phloem and vascular developmental processes during the fiber maturation in the ramie stem, and ethylene and gibberellin are likely to be involved in this process. Our findings provide novel insights into the phloem development and fiber maturation in ramie, which could be useful for fiber improvement in ramie and other fiber crops.