OTUB1 is a key factor of the estrogen signaling pathway in mammals. It has been proved that OTUB1 is a novel ERα-interacting protein with capability of deubiquitinating ERα and then repressing its transcriptional activity. In this study, expressed sequence tag (EST) of Scylla paramamosain OTUB gene(OTU-domain Ub-aldehyde-binding protein)was identified from transcriptome sequencing library and cloned by using SMART Rapid Amplication of cDNA end (RACE) technique. The full-length cDNA of Sp-OTUB is 1 261 bp, including 40 bp in 5'-UTR, 804 bp in open-reading frame (ORF) and 417 bp in 3'-UTR. The ORF encodes 365 amino acids with a predicted molecular weight of 31 ku, and the translated protein contains a typical OTU-like cysteine protease catalytic domain and a putative catalytic triad which was composed of conserved cysteine, histidine, and possibly the aspartate residues. Real-time quantitative PCR showed that Sp-OTUB was expressed in all detected tissues of mature crabs with the highest expression in haemolymph and the lowest expression in testis. In the process of ovarian development, Sp-OTUB showed the highest expression in proliferation stage compared to other development stages. In secondary spermatocyte stage, the expression of Sp-OTUB reached the highest and was significantly higher than that of primary spermatocyte stage or spermatid stage. Phylogenetic analysis based on the OTUB protein homologue sequences indicated that Sp-OTUB was separated together with OTUB1 in most other species, and Sp-OTUB had the closest genetic relationship with that of Ixodes scapularis or Strongylocentrotus purpuratus. The preliminary experimental results showed that Sp-OTUB may play an important role in reproductive regulation.