Astaxanthin, a high-value red ketocarotenoid with strong antioxidant activity, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis can accumulate up to 4% of fatty acid-esterified astaxanthin (by dry weight) and has been used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although the coordination of astaxanthin and fatty acids biosynthesis in a stoichiometric fashion was observed in H. pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying the coordination between the two pathways in H. pluvialis. Our results eliminated the possible coordination of this interdependence at the transcriptional level, and proved that this interaction was feedback related at the metabolite level. The in vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further proved that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H. pluvialis was subsequently proposed. These findings provided further insights into astaxanthin biosynthesis in H. pluvialis.This article is protected by copyright. All rights reserved.