Abstract: This study aimed to unravel whether DGATs are involved in the biological processes of astaxanthin acylation in H. pluvialis. A type II DGAT-encoding gene, DGTT2, was identified in H. pluvialis NIES-144 through RNA-seq and cloned from the cDNA library. Overexpression of HpDGTT2 in the TAG-deficient Saccharomyces cerevisiae strain H1246 failed to rescue the TAG-deficient phenotype, indicating that HpDGTT2 cannot utilize diacylglycerol for TAG biosynthesis. Meanwhile, an in vitro enzymatic assay system for measuring astaxanthin:acyl-CoA acyltransferase activity was established successfully by using H. pluvialis endoplasmic reticulum membranes as crude enzymes. When the S. cerevisiae microsomes containing recombinant HpDGTT2 were incorporated into the system, the concentrations of a number of astaxanthin esters increased significantly compared with the control, which indicated that HpDGTT2 possesses astaxanthin acyltransferase activity. This study will provide a basis for further studies on the function and metabolic regulation of HpDGTT2 in astaxanthin biosynthesis.