Abstract: In this research, the effects of different concentrations of melatonin (MLT) on the growth, accumulation of astaxanthin, reactive oxygen species (ROS), signal molecules, and the expression of DXS were studied in Haematococcus pluvialis LUGU under abiotic stress conditions (e.g., high light, nitrogen starvation, etc.). The results showed that the astaxanthin content was efficiently enhanced in alga cells treated with 10 μmol/L of MLT, the maximum astaxanthin content achieved was 31.32 mg/g, being 2.36 times higher than that of the control. In addition, MLT inhibited intracellular ROS levels and up-regulated the content of signaling molecules (nitric oxide and salicylic acid). Additionally, the relative expression level of DXS gene was increased by 11.3-fold higher than that of control. These results demonstrated that the massive accumulation of astaxanthin in Haematococcus pluvialis is associated with the regulation of intracellular ROS, signaling molecules, and gene expression by exogenous MLT under abiotic stress conditions.