Background <p>The color of microalgae is related to the components and contents of pigments. Yellow microalgae have a low chlorophyll content, which is helpful for obtaining high-purity lutein during downstream extraction. Chlorophyll and lutein content changes under different culture modes if chlorophyll biosynthesis is not fully destroyed. Low chlorophyll content improved the purity of the final lutein product. It is not fully known about the lutein biosynthesis and pigment dynamics in yellow microalgae under different culture modes.</p> Results <p>A yellow algal mutant <i>Chlorella</i> sp. M04 strain was obtained via chemical mutagenesis. The effects of the culture mode (autotrophic, mixotrophic, and heterotrophic) and carbon source were evaluated on the original strain (<i>Chlorella</i> sp. AE10), and the mutant. The ratios of lutein contents to (Chl a + Chl b) contents in the wild type and the mutant were 1:3.5 and 1:0.79, respectively. The highest lutein production observed in the original strain was 3.87&#xa0;mg/L/d at day 3 under mixotrophic conditions. The lutein productivity of the mutant under heterotrophic conditions was 1.88&#xa0;mg/L/d on day 2, which was higher than that under autotrophic and mixotrophic conditions. The chlorophyll-a content of the mutant (<i>Chlorella</i> sp. M04) decreased by 88.8% of chlorophyll-a content of the original strain, and chlorophyll b of the mutant was 7.2% of that of the original one under mixotrophic conditions on day 5. Glucose was found to be more suitable than acetic acid for the mutant. Comparative transcriptomic analysis was performed for the original and mutant strains, particularly for the biosynthesis pathways of chlorophyll and lutein.</p> Conclusion <p>The advantage of this mutant is its low chlorophyll content, making it suitable for downstream processes. This study provides new insights into the metabolic regulation of chlorophyll and lutein production in microalgae.</p> Graphical Abstract <p></p>