In this study, full-length cDNA of α-tubulin in sea cucumber Apostichopus japonicus was cloned for the first time by transcriptome data mining and rapid amplification of cDNA ends (RACE), and qRT-PCR was used to preliminarily reveal the biology function of α-tubulin in the process of intestine regeneration. The results showed that full-length cDNA of α-tubulin was 1641 bp, containing a 1362 bp open reading frame encoding a putative polypeptide of 453 amino acids residues. Bioinformatics analysis showed that the sequence of α-tubulin contains a 5'-untranslated region (UTR) of 153 bp, and a 3'-UTR of 126 bp. The predicted molecular mass of the deduced amino acid of α-tubulin was 50.33 ku, and the theoretical isoelectric point was 4.89. The amino acids inferred have a special sequence (GGGTGSG) which was a typical element in α-tubulin protein, belonging non-transmembrane and hydrophilic protein. Multiple sequence alignment analysis and phylogenetic analysis revealed that the α-tubulin in sea cucumber was very conservative with other eukaryotic organism and had 90% sequence identity with Notothenia coriiceps. Real-time quantitative PCR was carried out to measure the expression level of α-tubulin gene in different stages of sea cucumber intestine regeneration. The expression of α-tubulin was detected at all stages of intestine regeneration. The maximum and the minimum expression levels were found on 17^th and 5^th day, respectively. This study first obtained the structure information of α-tubulin gene in sea cucumber, and further certified the high homology of α-tubulin in eukaryote. In addition, our results indicated that α-tubulin plays an important role during intestinal regeneration in the sea cucumber A. japonicus.