Current mixotrophic culture systems for Dunaliella salina have technical limitations to achieve high growth and productivity. The purpose of this study was to optimize the mixotrophic conditions imposed by glycerol, light, and salinity that lead to the highest biomass and β-carotene yields in D. salina. The combination of 12.5 mM glycerol, 3.0 M salinity, and 50 μmol photons m-2 s-1 light intensity enabled significant assimilation of glycerol by D. salina and consequently enhanced growth (2.1 × 106 cell mL-1) and β-carotene accumulation (4.43 pg cell-1). The saline and light shock induced the assimilation of glycerol by this microalga. At last stage of growth, the increase in light intensity (300 μmol photons m-2 s-1) caused the β-carotene to reach values higher than 30 pg cell-1 and tripled the β-carotene values obtained from photoautotrophic cultures using the same light intensity. Increasing the salt concentration from 1.5 to 3.0 M NaCl (non-isosmotic salinity) produced higher growth and microalgal β-carotene than the isosmotic salinity 3.0 M NaCl. The mixotrophic strategy developed in this work is evidenced in the metabolic capability of D. salina to use both photosynthesis and organic carbon, viz., glycerol that leads to higher biomass and β-carotene productivity than that of an either phototrophic or heterotrophic process alone. The findings provide insights into the key role of exogenous glycerol with a strategic combination of salinity and light, which evidenced unknown roles of this polyol other than that in osmoregulation, mainly on the growth, pigment accumulation, and carotenogenesis of D. salina.