The Chinese mitten crab (Eriocheir japonica sinensis) is one of the most popular breeding species of crab in China due to its high economic and nutritional value. However, earlier published genome assemblies of E. j. sinensis have been highly fragmented due to limited sequencing and assembly techniques. In this study, we performed high-coverage sequencing of the Chinese mitten crab using advanced high-through chromosome conformation capture (Hi-C) technology and anchored these Hi-C data to previously published contig-level genome assembly for chromosome construction. Thus, we successfully assembled the first chromosome-level reference genome of E. j. sinensis, which contained 72 chromosomes with a N50 length of 17.61 Mb. Evaluation results demonstrated a high degree of genomic connectivity and accuracy. Acquisition of the chromosome-level genome of E. j. sinensis not only provides a basis for the study of adaptive evolution in crabs by comparative genomics, but should also help in studies on the growth, development, and reproduction of the Chinese mitten crab.