Abstract: At present, there are few researches on the pearl breeding mechanism of the high-quality freshwater mussel Hyriopsis schlegelii, and the Pif gene is closely related to the formation of shellfish pearls. In order to explore the preliminary function of Pif gene of Hyriopsis schlegelii, the full-length cDNA sequence of Pif gene was obtained for the first time in Hyriopsis schlegelii by RACE-PCR technique, which is named HsPif. The full-length untranslated region (5'UTR) of 5'- terminal untranslated region of Pif gene was 485 bp, 3' untranslated region (3'UTR) and 363 bp, open reading frame (ORF) 3072 bp, which encodes a total of 1023 amino acids. It was speculated that the bioinformatics analysis of two proteins encoding HsPif97 and HsPif80 showed that HsPif protein contained one von-Willebrand factor A (VWA) domain and three chitin binding domains (ChtBD2). The results of amino acid composition showed that the content of aspartic acid was the highest, accounting for 12.5%, and the content of histidine was the lowest, accounting for 1.32%. The α helix content in the protein secondary structure accounted for 19.63%, the irregular crimp accounted for 55.09%, and the extension chain accounted for 16.86%. The hydrophilic index of HsPif protein was –0.566. Phylogenetic tree analysis showed that HsPif and Hyriopsis cumingii Pif were the most conservative in one branch and on the same large branch as other shellfish. The results of tissue fluorescence quantitative PCR showed that HsPif gene was mainly expressed in the mantle, and in situ hybridization showed that the hybridization reaction mainly occurred in the mantle epithelial cells. The changes of HsPif gene expression were detected by qPCR after grafting. The results showed that HsPif gene expression changed with the development of pearl sac. The expression level of HsPif gene decreased significantly on the 7th and 90th day after nuclear insertion, and significantly increased on the 15th, 60th and 120th day. There was no significant change between the 30th day and the control group. The above experimental results suggest that HsPif gene may be related to the secretion and formation of pearls, which is helpful to further understand the mechanism of pearl formation and provide reference for pearl culture.