Portunus trituberculatus is the primary marine crab species cultured in China. Molting is vital for growth,reproduction, and regeneration in crustaceans. Molting is regulated by a complex molecular mechanism but the detailsremain unclear and require further research. Calmodulin (CaM) is a conserved multifunctional protein that regulatesCa2+ and glycogen metabolism, influences cell division and movement, and is involved with neurotransmitter synthesisand release. Choosing and cloning a CaM gene to investigate the functions and roles of CaM during molting depends onthe transcriptome data. P. trituberculatus CaM cDNA was cloned using rapid amplification of cDNA ends and namedPTCaM. The PTCaM full-length cDNA was 1981 bp, including a 450-bp open reading frame that encoded a 256 aminoacid polypeptide, with a molecular mass of 16.8 kD and an isoelectric point of 4.09. A bioinformatics analysis revealed thatPTCaM has a highly conserved sequence with a typical Ca2+ EF-hand binding domain of the EF superfamily. The EF-handis a calcium binding motif with two active canonical EF hands. Ca2+ binding induces a conformational change in theEF-hand motif leading to activation or inactivation of target proteins. A homology analysis showed that PTCaM had thehighest homology with Eriocheir sinensis and Drosophila melanogaster. A phylogenetic analysis showed that PTCaM wasin the same branch with Litopenaeus vannamei, E. sinensis, and Procambarus clarkia. A real-time quantitative polymerasechain reaction analysis of different molting stages showed that PTCaM was expressed significantly differently in all tissuestested during molting, which reveals a molting function for PTCaM. PTCaM expression in the eyestalk and gill weredownregulated during the post-molt stage, suggesting that PTCaM may participate in preparation for molting. PTCaMexpression was upregulated in heart, hemocytes, and muscle during the pre-molt stage, suggesting that PTCaM may regulateion transport, particularly that of Ca2+. PTCaM expression was downregulated in most tissues after eyestalk ablation,compared to that in normal control tissues. PTCaM may cooperate with molt inhibiting hormone or ecdysone because single-eyestalk ablation has less of an inhibiting effect on ecdysone synthesis.