Abstract: 【Objective】To clarify the sequence characteristics， tissue expression rules and its expression patterns in response to acute heat stress of citrate synthase（CS）gene（SiCS）in Strongylocentrotus intermedius. The results of this study would provide a theoretical basis for further study of the biological function of SiCS gene and reveal the molecular mechanism of its involvement in the high temperature response of S. intermedius.【Method】The cDNA sequence was cloned by using the rapid amplification of cDNA ends（RACE）technology. Bioinformatics analysis was carried out by online softwares such as BLASTx，EXPASY Proteomics Server，HMMER，PSIRED v3.3 and SWISS-MODEL. Mitochondria swelling degree of the tube foot and gonad were measured by spectrophotometry. Tissue relative expression of SiCS was analyzed by real-time fluorescence quantitative PCR（qRT-PCR）；the changes of total SiCS activity in the tube foot and gonad of S. intermedius were measured by Epoch assay.【Result】The results showed that the full length of SiCSc DNA was 2971 bp，containing a 5' end non-coding region（5'-UTR）of 96 bp，a 3' end non-coding region（3'-UTR） of 1480 bp， and an open reading frame （ORF）of 1395 bp which encoded 464 amino acids residues. The predicted molecular weight and the theoretical isoelectric point（pI）of SiCS protein were 51.48 kD and 6.09 respectively. SiCS was a stable mild hydrophobic protein with an average hydrophilicity（GRAVY）of-0.178. SiCS had no signal peptideand transmembrane. In the secondary structure，α-helix accounted for 43.86%，β-fold accounted for 7.02%，and random coil accounted for 49.12%.The consistency between the three dimensional structure of SiCS and the crystal structure of CS protein from Sus scrofa was 73.97%. Phylogenetic analysis indicated that the SiCS protein had the most similarity with CS protein from Strongylocentrotus purpuratus（similarity： 92.90%）. Relative gene expression data showed that SiCS was expressed in all examined tissues and the expression level from high to low was as coelomcytes>interdental muscle>gonad>tube foot>intestine. Total enzyme activity assay revealed that the level of total SiCS enzyme activity in different tissues from high to low was as tube foot>intestine>interdental muscle>gonad>coelomcytes. Compared with the control group（20 ℃），the relative expression level of the SiCS gene and the total SiCS activity in the tube foot and gonad of S. intermediuss howed fluctuations after acute heat stress at 23 and 26 ℃，and there were some differences under different heat stresses and in different tissues.【Conclusion】S. intermedius mightrespond to acute heat stress by adjusting the relative expression of SiCS gene and SiCS enzyme activity in different tissues， and this response strategy hascertain tissue and temperature specificity，which specifically shows that the gonad is more sensitive to acute heat stress than the tube foot.