In order to study the role of another Cathepsin L (CtsL) isoform from Hyriopsis schlegelii in immune responses, we isolated and characterized a novel CtsL gene from the freshwater pearl mussel Hyriopsis schlegelii using cDNA library screening and rapid amplification of cDNA ends (RACE), and it was named HsCtsL1-like (GenBank accession No. KF015273). The HsCtsL1-like cDNA was 1280 bp long, and consisted of a 5'-untranslated region (UTR) of 31 bp, a 3'-UTR of 256 bp with a polyadenylation signals (AATAAA) at 11 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 993 bp encoding a polypeptide of 330 amino acids with 36.86 ku predicted molecular weight. The theoretical isoelectric point was 6.23. Sequence analysis showed that the putative HsCtsL1-like protein sequence sharing similar structural features with other reported CtsLs containing a typical signal peptide sequence with 16 amino acids (Met¹~Ser¹⁶), a propeptide inhibitor domain(Try²⁵~Phe⁸⁴) and a mature domain(Leu¹¹⁴~Val³³⁰). And some signature sequence tags were identified for CtsL family proteins in other species including the oxyanion hole (Gln), the active triad formed by Cys, His and Asn, and the conserved ERF/WNIN, GNFD, GCXGG and OCHN motifs can all be found in the HsCtsL1-like protein sequence. Moreover, homology analysis revealed that HsCtsL1-like shared the highest identity (67%) with CtsL1 (AGL33704.1) from the razor clam, Sinonovacula constricta, while the similarities of HsCtsL1-like with the reported Hyriopsis cumingii (ADV03094) and H. schlegelii CtsL (AEX88474) were both 52.91%. The phylogenetic tree revealed that the HsCtsL1-like clustered with the invertebrate CtsL cysteine proteases and was also closely related to S. constricta, Crassostrea gigas and Pinctada fucata CtsL1. Real-time quantitative reverse transcription PCR (qRT-PCR) showed that HsCtsL1-like is expressed in a wide range of tissues including the liver, gills, foot, heart, ovary, testis, kidney, blood, intestines and mantle, with the highest level of transcripts in liver, followed by ovary, testis, intestines and blood, while there is rare expression in other tissues. After Vibrio anguillarum stimulation, the expression level of HsCtsL1-like mRNA was significantly up-regulated at 2 h and 8 h in liver and blood. These results suggested that HsCtsL1-like belongs to subtype 1 of the CtsL subfamily, and it might play an important role in the freshwater mussel, H. schlegelii immune response.