Simple SummaryTheFem-1cgene inEriocheir sinensis(Henri Milne Edwards, 1854) (EsFem-1c) is related to sex differentiation; however, its functional mechanism is less reported. In this study, qRT-PCR results ofEsFem-1cfrom the intersex crab implied thatEsFem-1cplays a role in crab androgenic gland (AG) development. RNAi and scanning electron microscope observations revealed thatEsFem-1cinfluenced sexual development inE. sinensis. A dual-luciferase reporter assay demonstrated that tcf-miR-315-5p binding to the 3′UTR sequence effectively inhibited the translation of theEsFem-1cgene, and tcf-miR-307 binding to the alternative splicing region in the 3′UTR sequence could increaseEsFem-1cexpression. In summary, this study provides a better understanding of the molecular regulation mechanism ofEsFem-1c.AbstractFeminization-1c(Fem-1c) is important for sex differentiation in the model organismCaenorhabditis elegans. In our previous study, the basic molecular characteristics of theFem-1cgene (EsFem-1c) inEriocheir sinensis(Henri Milne Edwards, 1854) were cloned to determine the relationship with sex differentiation. In this study, the genomic sequence ofEsFem-1ccontained five exons and four introns, with an exceptionally long 3′UTR sequence. The qRT-PCR results ofEsFem-1cdemonstrated lower tissue expression in the androgenic gland of the intersex crab than the normal male crab, implying thatEsFem-1cplays a role in crab AG development. RNA interference experiments and morphological observations of juvenile and mature crabs indicated thatEsFem-1cinfluences sexual development inE. sinensis. A dual-luciferase reporter assay disclosed that tcf-miR-315-5p effectively inhibits the translation of theEsFem-1cgene, influencing male development. An intriguing finding was that miRNA tcf-miR-307 could increaseEsFem-1cexpression by binding to the alternative splicing region with a length of 248 bp (ASR-248) in the 3′UTR sequence. The present research contributes to a better understanding of the molecular regulation mechanism ofEsFem-1cand provides a resource for future studies of the miRNA-mediated regulation of sexual development and regulation inE. sinensis.