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<i>Chromochloris zofingiensis</i>, a unicellular green alga, is a potential source of natural carotenoids. In this study, the mutant LUT-4 was acquired from the chemical mutagenesis pool of <i>C. zofingiensis</i> strain. The biomass yield and lutein content of LUT-4 reached 9.23 g·L⁻¹, and 0.209% of dry weight (DW) on Day 3, which was 49.4%, and 33% higher than that of wild-type (WT), respectively. The biomass yields of LUT-4 under 100, 300, and 500 µmol/m²/s reached 8.4 g·L⁻¹, 7.75 g·L⁻¹, and 6.6 g·L⁻¹, which was 10.4%, 21%, and 29.6% lower compared with the control, respectively. Under mixotrophic conditions, the lutein yields were significantly higher than that obtained in the control. The light intensity of 300 µmol/m²/s was optimal for lutein biosynthesis and the content of lutein reached 0.294% of DW on Day 3, which was 40.7% more than that of the control. When LUT-4 was grown under 300 µmol/m²/s, a significant increase in expression of genes implicated in lutein biosynthesis, including phytoene synthase (<i>PSY</i>), phytoene desaturase (<i>PDS</i>), and lycopene epsilon cyclase (<i>LCYe</i>) was observed. The changes in biochemical composition, Ace-CoA, pyruvate, isopentenyl pyrophosphate (IPP), and geranylgeranyl diphosphate (GGPP) contents during lutein biosynthesis were caused by utilization of organic carbon. It was thereby concluded that 300 µmol/m²/s was the optimal culture light intensity for the mutant LUT-4 to synthesize lutein. The results would be helpful for the large-scale production of lutein.