CYP302a1 is the key enzyme in biosynthesis of the ecdysteriods. The aim of this study is to analyze the function of CYP302a1 during the process of the molting in swimming crab Portunus trituberculatus. The full-length cDNA of CYP302a1 is cloned from Y-organs of the P. tritubertulatus. The length of the CYP302a1(Genbank accession NO.: KM596851)is 3 171 bp and contains an ORF of 1 626 bp encoding 541 amino acid. The amino acid contains 5 conserved domains including helix-C, helix-K, helix-I, PERF and heme-binding. The phylogenetic tree showed that CYP302a1 of P. trituberculatus was clustered with that of Tigriopus japonicas, which is separated from other CYPs. Tissue distribution showed that transcript of CYP302a1 was mainly detected in the Y-organ(YO)and far exceeded other tissues. The transcript level of CYP302a1 during molting cycle was determined by qRT-PCR. During molting process, levels of CYP302a1 in YO were extremely low during postmolt(stages A and B), began to increase during intermolt(stage C)and reached the peak at D₁ stage, then declined. The combined results showed that CYP302a1 might play an essential role in the molting of P. trituberculatus.