The molting process is an essential physiological process in crustaceans that is closely related to the synthesis of ecdysteriods. Cytochrome P450(CYP)302a1 is the key enzyme which plays a critical role in the synthesis of ecdysteriods. Here we present the cloning and characterization of CYP302a1 gene from Macrobrachium rosenbergii (Mr-CYP302a1). The acquired gene was 1 859 bp in full-length with the open reading frame (ORF) of 1 629 bp that encodes 543 amino acids (aa) with a molecular weight of 61.09 ku and an isoelectric point of 8.42. The aa sequence analysis revealed that there were five P450 characteristic conserved regions, i.e., heme-binding, helix-K, helix-C, helix-I, and PERF. Phylogenetic analysis demonstrated that Mr-CYP302a1 was closely related to the CYP302a1 of Neocaridina denticulata, and then clustered with the CYP302a1 from Decapoda crustaceans such as Litopenaeus vannamei and Portunus trituberculatus. Real-time quantitative PCR (qRT-PCR) results showed that Mr-CYP302a1 was expressed in almost all the tissues tested with significantly higher expression levels in the Y-organ. On the other hand, the expression of Mr-CYP302a1 was significantly lower at the postmolt stage (stages A and B), and it was increased gradually at the intermolt (stage C), significantly enhanced and reached the maximal level at the D₁ stage. Mr-CYP302a1 was expressed and its polyclonal antibody was generated. Western blot (WB) showed that the expression of Mr-CYP302a1 protein was the highest in Y- organs of M. rosenbergii. The expression level of Mr-CYP302a1 protein also reached a peak at D₁ stage during the molting process. In summary, our results indicate that Mr-CYP302a1 may play an important role in molting of M. rosenbergii.