Pearl oyster Pinctada fucata is the most popular farming shellfish for seawater pearl production in Guangdong，Guangxi and Hainan province of China. In recent years，as other marine animals，higher frequencies of disease epidemics and the emergence of new diseases have been reported in artificial cultivation of Pearl oyster. Many researchers considered the reasons for high mortality were ocean pollution， disease outbreaks and stock degeneration. In order to control disease and enhance the yields and quality of seawater pearl，it is necessary to further research the innate immune mechanisms of pearl oyster. Cathepsin L is a member of cysteine protease family and involved in various biological functions，which is distributed widely in living organisms. In this study，we identified one cDNA encoding a cathepsin L cysteine proteases from EST library of pearl oyster Pinctada fucata （designated as poCL2）. The poCL2 cDNA was 1 094 bp long and consisted of a 5 ′ -untranslated region （UTR） of 21 bp，a 3 ′ -UTR of 80 bp with a polyadenylation signal （AATAAA），and an open reading frame （ORF） of 993 bp encoding a polypeptide of 330 amino acids，which contained a typical signal peptide sequence （Met¹-Ala¹⁶）、a prodomain （Arg¹⁷-Asp¹¹³），and a mature domain （Leu¹¹⁴-Val³³⁰）. The preprocathepsin contained a potential N-glycosylation site （Asn⁹⁷），six substrate binding sites （Leu¹⁸²，Met¹⁸³，Ala²⁴⁹，Leu²⁷⁵，Gly²⁷⁸ and Ser³²⁴），three catalytic sites （Cys¹¹³，His²⁷⁸，and Asn²⁹⁸）. The conserved E⁴²X₃RX₃WX₂NX₃IX₃N⁶¹ motif is discovered in the prodomain which may be play important function in the inhibition of proteolytic activity. The G⁷⁴X₁NX₁YX₁D⁸⁰，which may be related to pH-dependent intramolecular processing，is also found in the prodomain of the poCL2. Position of prodomain cleavage site and conserved cysteine residues are based on the N-terminal sequence information from HumanCL1 （Homo sapiens，P07711），RatCL1 （Rattus norvegicus，P07154），BovineCS （Bos taurus，P25326），and ChickenCL （Gallus gallus，P09648），and the prodomain cleavage site was between Asp113 and Leu114.The number of N-glycosylation site is not conserved，there is one，two and three potential N-glycosylation sites in Ictalurus punctatus，Toxoplasma gondiis and Trypanosoma carassii cathepsin L，respectively，which revealed that the intracellular transport mechanism of cathepsin L proteases is different in living organisms. Homology analysis of poCL2 by MatGAT software revealed that the poCL2 shared 61.5-73.9% similarity and 44.4-59.8% identity to other known cathepsin L sequences. The phylogenetic tree showed that the poCL2 clustered with the invertebrate cathepsin L cysteine proteases and was closely related to Radix peregra cathepsin L，and a clear clade division was observed between vertebrates and invertebrates cathepsin L proteins. The previous study demonstrated that the expression pattern of cathepsin L was different in different species and exhibited specificity for some tissues in different species. The mRNA expression of the poCL2 in normal group could be detected in digestive gland，gonad，haemocytes，gills，mantle，adduct muscle and intestines，and with the higher level in mantle，the results indicated that the expression pattern of cathepsin L gene in various tissues was somewhat different，and had diverse functions. The digestive gland of mollusk was thought to be an important immune organ，which could secrete various enzymes to hydrolyze microorganisms and involve in digestive and defense functions. So we selected the digestive gland to research the temporal expression pattern of poCL2 after Vibrio alginolyticus and LPS challenge. The results showed that the expression level of the poCL2 was up-regulated in digestive gland not only in V. alginolyticus group but also in LPS group，and the highest expression was observed at 8 h after stimulation and was 1.73-fold，1.25-fold higher than the control group，respectively. These results suggested that the poCL2 was involved in the innate immune response of pearl oyster and might play an important function in immune regulation.