Abstract: Chromochloris zofingiensis can achieve ultra-high cell density and simultaneously accumulate astaxanthin under heterotrophic conditions, however, the relatively low cellular astaxanthin content of C. zofingiensis hinders its commercial application. With the aim to improve the ellular astaxanthin content of C. zofingiensis, this study screened a mutant 12C10 with enhanced astaxanthin content from the mutant library established by using ethylmethylsulfone mutagenesis. The astaxanthin content of 12C10 was 74% higher than that of the wild type under the heterotrophic normal conditions, and 25% higher than that of the wild type at day 4 under the nitrogen deficiency conditions. Additionally, the results of the widely targeted metabolomics analysis revealed the differences in the metabolites level between 12C10 and wild type under the normal conditions. Specifically, the contents of fatty acids and amino acids except glutamate generally decreased, but the content of glutamate significantly increased in 12C10 compared to wild type. Reduced synthesis of amino acids and fatty acids in 12C10 can provide more carbon skeleton, NADPH, and ATP for astaxanthin synthesis. The accumulation of glutamate on the one hand may stimulate the activity of glucose 6-phosphate dehydrogenase in pentose-phosphate pathway to produce more NADPH, and cause production of reactive oxygen species to trigger astaxanthin synthesis. The metabolome analysis also showed that the enhanced astaxanthin synthesis in 12C10 may be related to the enhanced ethylene synthesis. This study lays the foundation for improving C. zofingiensis astaxanthin content through metabolic regulation in the future, and is of great significance for guiding the development of the new processes of C. zofingiensis astaxanthin accumulation.