Xanthophyll esters are present in a large number of plants and microalgae, but its biosynthesis process is poorly understood. We demonstrated that acylation of xanthophylls is catalyzed by type I diacylglycerol acyltransferase (DGAT) from the unicellular green alga Haematococcus pluvialis. Expression of HpDGAT1, and three of the four HpDGAT2 genes showed significant up-regulation under all the three adverse environmental conditions to trigger astaxanthin esters biosynthesis, respectively. Addition of recombinant HpDGAT1 into the enzymatic assay system with the endoplasmic reticulum membranes of H. pluvialis as the enzyme sources further significantly increased net xanthophyll esters formation. Overexpressing HpDGAT1 in an engineered Escherichia coli strain capable of producing free astaxanthin led to enhanced accumulation of both free astaxanthin and xanthophyll esters with maximum UV absorption at 480 nm, the typical feature of astaxanthin esters. Application of DGAT1 specific inhibitor A922500 decreased the content of astaxanthin esters up to 36% in H. pluvialis under high light stress. These data suggested that HpDGAT1 act as xanthophyll acyltransferase in H. pluvialis. Our findings broaden the understanding of xanthophylls acylation in microalgae.