The eukaryotic initiation factor 4E protein (4E-BP1) is the main downstream factor of the mTOR signaling pathway. It is phosphorylated by an activated mTORC1 signal and acts as a translation inhibitor. 4E-BP1 plays a key role in the rate-limiting of cap-dependent translation, thus influencing protein synthesis, cell survival, growth, proliferation, and metabolism. Although the regulatory mechanism of 4E-BP1 has been well elucidated in mammals, the knowledge regarding crustaceans is still limited. In this study, we obtained a full-length cDNA of Eriocheir sinensis 4E-BP1 (Es4E-BP1) using the RCAE method. Real-time quantitative PCR (qPCR) was applied to detect the temporal and spatial expression patterns of Es4E-BP1. The double-stranded RNA (dsRNA) silencing method was used to explore the role of Es4E-BP1 in the molting cycle of E. sinensis. Results showed that the length of Es4E-BP1 cDNA was 1678 bp, containing a 134 bp 5′ non-coding region, 1193 bp 3? non-coding region, and 351 bp open reading frame (ORF). The cDNA sequence is composed of three exons (282 bp, 165 bp, and 1209 bp) and two introns (2101 bp and 622 bp). The ORF encodes a putative polypeptide of 116 amino acids with a molecular weight of 12.64 kD and an isoelectric point (PI) of 4.89. The secondary structure of Es4E-BP1 contains a typical eIF4E superfamily conserved domain, a TOR signaling motif, and a Y××××LΦ binding motif (Φ denotes any hydrophobic amino acid), which are unique to the 4E-BPs family. Multiple sequence alignment and phylogenetic tree analysis showed that Es4E-BP1 was clustered with Callinectes sapidus and Portunus trituberculatu, with high identities of 95.69% and 93.16%, respectively. qPCR results showed that Es4E-BP1 was expressed in various tissues of adult crabs. The transcript level in Y organs was significantly higher than that in other tissues, such as muscle, heart, eyestalk, intestine, ovary, testis, hepatopancreas, and gill (P＜0.05). During a complete molting cycle, the expression patterns were distinct among different tissues of juvenile crabs. There is a similar expression pattern in Y-organ and intestine. The highest expression levels were observed in post-molt (stage A-B), followed by intermolt (stage C) and early post-molt (stage D₀ and stage D₁). The expression levels were the lowest in the post-molt (stage D_3-4). The expression level in the eye stalks was the highest in stage A–B. It decreased in stages C and D0 but increased in stage D1 and stage D_3-4. In addition, the expression level of Es4E-BP1 differed among different muscle tissues. The expression levels of Es4E-BP1 in the claw muscle and abdominal muscle reached the peak in stage A–B, was the lowest in stage C (P＜0.05), and increased slightly in stage D_3-4 compared with stage C. In the walking foot muscle, the expression levels were high in stages A–B and C but decreased in stage D_3-4 (P＜0.05). The RNA interference test showed that the mRNA levels of Es4E-BP1 were significantly decreased in the Y-organ and muscle after injection with ds4E-BP1. In the Y-organ, the expression level of Es4E-BP1 in the injected group was 53.77% (24 h post-injection) and 41.99% (48 h post-injection) of that in the control group. Meanwhile, in muscles, the expression level of Es4E-BP1 in the injection group was 44.00% (24 h post-injection) and 27.49% (48 h post-injection) of that in the control group. Furthermore, during the long-term interference experiment, dsEs4E-BP1 was injected every 2 days. The survival rate of the ds4E-BP1 injection group was 38.89%, much lower than 61.11% in the control group and 50.00% in the dsGFP injection group. The average molting interval of the ds4E-BP1 injection group was 38.29 days, much longer than that of the control group (34.45 days) and dsGFP injection group (33.10 days). The weight gain rate of the ds4E-BP1 injection group was 34.32%, higher than that in the control group (28.69%) and dsGFP injection group (26.95%). Overall, our results indicated that Es4E-BP1 might play a crucial role in the molting and growth of E. sinensis.