In order to study the role of the isoamylase gene (ISA3) in the metabolism of yam tuber starch, we used Bikeqi yam and Dahechangyu yam as the measure to determine the starch and its component content and isoamylase (ISA) activity. The RTPCR technology has been used to clone the open reading frame of ISA3. The bioinformatics analysis were performed. Expression of ISA3 gene in different swelling stages tuber and among different tissues of yam was analyzed. The results showed that: (1) the amylose, amylopectin and total starch contents of Dahechangyu yam are significantly higher than Bikeqi yam, and the starch content of the two varieties showed a trend of first increase and then decrease with the change of growth and development, it reached the highest level at 120 d after planting. However, the ISA activity of Bikeqi yam was higher than that of Dahechangyu yam during the whole expansion period. (2) We successfully isolated an isoamylase gene (ISA3) from yam variety Bikeqi and Dahechangyu. Its sequence length is 1 584 bp and encodes 527 amino acids. ISA3 is a hydrophilic protein and nontransmembrane structure. (3) In different periods of yam tuber expansion of 2 varieties with different starch contents, ISA3 gene expression increases first and then decreases in Bikeqi yam, but gradually decreases in Dahechangyu yam. It is expressed in the leaves, stem and tuber, and there is obvious tissue specificity. (4) ISA activity was extremely significantly and significantly positively correlated with yam starch content and amylose content. Nevertheless, ISA activity was negatively correlated with the expression of ISA3. This result suggests that ISA is involved in the synthesis of starch in tubers and play a key role in the synthesis of amylose, and ISA3 expression may play negative regulation of ISA activity and starch synthesis.