During pond culture of Eriocheir sinensis, a high limb-impairment rate restricts the industry development and quality. Therefore, research on limb autotomy and regeneration has important practical significance for the industrial development and basic biology of E. sinensis. This study evaluated the changes in bud morphology, growth-related gene expression and nutritional status during cheliped regeneration in E. sinensis. The study found that the new cheliped was pre-formed in the bud and then regenerated with the completion of molting of E. sinensis. The new cheliped was similar in morphology to the normal cheliped after the first molting but smaller in size. The qRT-PCR results of growth-related genes showed that the expression levels of EcR-mRNA (ecdysteroid receptor) and Chi-mRNA (chitinase) were significantly up-regulated, whereas the expression of MIH-mRNA (molt-inhibiting hormone) was significantly down-regulated (P < 0.05). The nutritional status during the regeneration process showed that the hepatopancreas total lipid content decreased significantly within 28 days and was significantly lower in the autotomy group than in the control group at 14 d and 21 d (P < 0.05). The hepatopancreas fatty acid composition results showed that saturated fatty acids (SFA), highly unsaturated fatty acids (HUFA) and n-3/n-6 were significantly higher in the autotomy group than in the control group at 21 d (P < 0.05), whereas the ∑ n-6 PUFA and ∑ n-3 PUFA at 1 d and 7 d, and the monounsaturated fatty acid (MUFA) at 28 d in the autotomy group were significantly lower than in the control group (P < 0.05). Moreover, the levels of eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) showed that DHA was significantly lower at 7 d and significantly higher at 21 d in the autotomy group than in the control group (P < 0.05), whereas ARA and EPA were not significantly different between the two groups. Muscle L-tryptophan content was significantly lower at 1 d and significantly higher at 7 d in the autotomy group than in the control group (P < 0.05). These results indicate that during the cheliped regeneration process, crabs could accelerate molting and regeneration by regulating growth-related gene expression (e.g., EcR-mRNA and MIH-mRNA) and nutrient metabolism (e.g., lipid metabolism).