<p>Astaxanthin, a high-value carotenoid, is predominantly produced by the green microalga <i>Haematococcus pluvialis.</i> To enhance astaxanthin production efficiency, heterotrophic or sequential heterotrophic-phototrophic cultivation strategies have been proposed as alternatives to conventional phototrophic cultivation. However, due to the cell polymorphism of motile and immotile forms, it is essential to investigate the heterotrophic growth characteristics and nutrient requirements of <i>H. pluvialis</i>. This study focused on evaluating the growth performance, nutrient requirements, and astaxanthin synthesis potential of <i>H. pluvialis</i> under fed-batch heterotrophic cultivation with acetate as the primary substrate. The results demonstrated that heterotrophic growth is significantly influenced by cell type and nutrient composition. Using immotile cells as the inoculum and a nutrient-rich medium supplemented with acetic acid, the cell dry weight reached 12 g L⁻¹, with a cell density of 7 × 10⁶ cells mL⁻¹ and biomass productivity of 0.74 g L⁻¹ day⁻¹. This was notably higher in immotile cells compared to motile cells grown under similar conditions, whether with only acetic acid or acetic acid with macro nutrients supplementation. Furthermore, immotile cells from fed-batch heterotrophic cultivation could undergo repeated heterotrophic cycles, whereas motile cells did not exhibit this phenomenon. Heterotrophically cultivated <i>H. pluviali</i>s cells were capable of synthesizing astaxanthin upon nitrogen-deprived light induction. Cells grown in concentrated medium exhibited enhanced light-induce astaxanthin synthesis, achieving a maximum astaxanthin productivity of 10.22 mg L⁻¹ day⁻¹. This study highlights the importance of cell type and optimized nutrient composition in designing efficient heterotrophic cultivation strategies for <i>H. pluvialis</i> to maximize astaxanthin production.</p>