To study the response of Gibberellins Insensitive gene (AtGAI) from amaranth to gibberellin, we cloned GAI gene from Amaranthus tricolor L. ‘Dahong’ by RTPCR combined with RACE technology, which is named as AtGAI (GenBank accession number: MK049175).The results showed that: (1) the AtGAI gene contains an open reading frame of 1 818 bp that encoding 605 amino acids. (2) Bioinformatics analysis indicated that AtGAI belongs to GAI family containing two conserved domains of DELLA and GRAS. (3) Phylogenetic tree analysis showed that the AtGAI protein was relatively close to the GAI protein of Amaranthus hypochondriacus. (4) Microscopic observation and betalain content analysis showed that the betalain was mainly distributed in the epidermis and parenchyma cells around the vascular bundle sheath of cotyledons and hypocotyls, and the GA₃ concentration was negatively correlated with the betalain content. (5) qRTPCR analysis showed that GA₃ inhibited the expression of AtGAI, AmMYB1, AmaDODA and AmCYP76AD1 genes in cotyledons and hypocotyls. The results suggest that GA₃ may regulate the betalain metabolism by affecting the expression of AtGAI gene.