Every autumn, large numbers of Chinese mitten crabs, Eriocheir sinensis, are sold, creating a backlog in availability reducing their sales. Crabs require a period of controlled rearing prior to their sale, which alleviates this fall backlog. This means that it has become increasingly important to establish an effective method for the overwinter rearing of E. sinensis. This study was designed to evaluate changes in the growth, nutritional quality and the composition of the gut microbiota of adult E. sinensis following overwinter rearing. The results revealed that the hepatosomatic index (HIS) significantly decreased while the gonadosomatic index (GSI) increased after overwinter rearing (p < 0.05) for 80 days. Percentages of total saturated fatty acid (ΣSFA) and monounsaturated fatty acid (ΣMUFA) in the three edible tissues (muscle, gonad and hepatopancreas) decreased after 80 days of overwintering. In addition, the amino acid content in all the three edible tissues diminished additionally, with the reduction in proline being the most significant (p < 0.05). Analyses demonstrated that overwintering affected the overall microbial community composition, and elevated the abundance of Candidatus Bacilloplasma; however, these changes were not significant (p > 0.05). Therefore, our results provide a key reference standard for the overwinter rearing of E. sinensis.