In order to identify and characterize the lactate dehydrogenase (LDH) gene and investigate its expression and activity alterations in response to seawater acidification stress in sea urchins, we used database data mining and rapid amplification of cDNA ends (RACE) to identify a novel LDH gene in the sea urchin Strongylocentrotus intermedius (here designated SiLDH). The full-length cDNA of SiLDH was 1 499 bp, including a 1 017 bp open reading frame encoding 338 amino acid residues. Sequence analysis indicated that the predicted SiLDH protein was similar to the LDH homolog in the sea urchin S. purpuratus. Quantitative real-time PCR analysis showed that SiLDH was ubiquitously expressed in coelomic fluid, gonad, tube foot and intestine of healthy adult S. intermedius, with the highest level of expression identified in the gonad. Total LDH enzyme activity in different tissues of S. intermedius from high to low was as gonad > tubefoot > coelomic fluid > intestine. Both SiLDH mRNA expression profiles and total LDH enzyme activity were altered in the coelomic fluid, gonad, tube foot and intestine after 60-day seawater acidification treatments (Control:natural seawater; OA₁:△pH=-0.3; OA₂:△pH=-0.4; OA₃:△pH=-0.5). Our results provide more information about the characteristics and biological functions of the LDH homolog in sea urchins.