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Objective To characterize the stage-specific transcriptome of terminal erythroid differentiation in bone marrow of β-654 thalassemia mice. Methods Genotyping and blood smear Wright-Giemsa staining were carried out to identify β-654 thalassemia mice and wild-type littermates. Bone marrow cells of β-654 thalassemia and wild-type littermates were then sorted by flow cytometry. ProE, BasoE, PolyE and OrthoE erythroblasts were collected respectively for RNA-seq; The sequencing data were filtered and subjected to differential expression analysis and subsequent GO analysis. Finally, protein-protein interaction network and hub of the up-regulated genes were depicted using STRING database and Cytoscape software. Results The quantity of differentially expressed genes between the β-654 thalassemic and stage-matched control erythroid cells increased gradually during terminal erythroid differenti- ation. GO analysis showed that chaperone-mediated protein folding process was gradually activated in thalassemic erythroid cells. Specifically, oxidoreductase activity was increased at BasoE to PolyE stages, and ubiquitin protein ligase binding was enhanced from PolyE to OrthoE stages; Protein-protein interaction analysis showed that heat shock protein (Hsp) family members were at the core of the thalassemic erythroid network and that more Hsp members join the network at later stages. Conclusions The thalassemic terminal erythroid differentiation shows stage-specific transcriptome characteristics. Protein folding mediated by Hsp family members is gradually activated during the whole process of terminal erythroid differentiation, oxidoreductase activity is selectively enhanced at BasoE and PolyE stages, while ubiquitin mediated-protein degradation is dominant during PolyE and OrthoE stages.