Most natural astaxanthin is fatty acid-esterified in microalgae to prevent oxidation. However, the factors influencing astaxanthin esterification (AE) are poorly understood. In this study, obstacles to AE in Coelastrum sp. HA-1 were investigated. Only half of the astaxanthin molecules in HA-1 were esterified, but AE was stimulated with exogenous linoleic acid (LA) and ethanol treatment. Astaxanthin esters and total astaxanthin (TA) with exogenous LA were elevated to 3.82-fold and 2.18-fold of control levels, respectively. Treatment with 3% (v/v) ethanol enhanced transcription of the Δ12 fatty acid desaturase gene, which caused more oleic acid (OA) to be converted to LA. Furthermore, the contents of astaxanthin esters and TA were 2.42-fold and 1.61-fold control levels, respectively. These findings confirmed that AE was upregulated by increasing LA content. Thus, a large concentration of OA alone does not increase astaxanthin accumulation in HA-1, and a certain amount of LA was necessary for AE.