Crustacean female sex hormone (CFSH) plays a pivotal role in developing adult-specific female characteristics, specifically the mating and parental care system of the female blue crab, Callinectes sapidus, manifested after the puberty-terminal molt. Therefore, this study aimed to determine if there is a relationship between CFSH and the development of phenotypes specific to the female C. sapidus during its life cycle and if the continued presence of CFSH is required for developing adult-specific features during the prepuberty molt cycle. First, CasCFSH transcripts in females steadily increase, starting from crab stages 3-4 to prepuberty, except for a decrease at crab stages 15-16. The prepuberty females have the highest CasCFSH transcripts, with ∼5.6-fold higher than adults. CFSH protein levels at prepuberty are the highest but similar to adults. The first and visible secondary female characteristics include a pair of gonopores or a triangular abdomen and four pairs of pleopods seen as early as crab stages at ∼3 (carapace width, CW, 5-7 mm) - 5 (CW 8-10 mm), respectively. The adult-specific female morphological features that are developed gradually throughout the prepuberty molt cycle are manifested only after the puberty-terminal molt. A CasCFSH knockdown study by injecting CasCFSH-dsRNA into prepuberty females at specific molt stages provides further evidence: the continued presence of CasCFSH is required for gradually developing adult-specific morphological features during the prepuberty molt cycle. Overall, the abdomen size is first determined at the intermolt stage. Then, the abdominal shape, gonopores, sternal tubercles, spermathecae, and ovigerous setae and hairs are formed at the intermolt and early premolt stages. Finally, the plumose hairs are developed during intermolt, early, and late premolt stages.