Dunaliella salina (Dunal) Teod is an extremely halotolerant unicellular eukaryotic alga whose salt tolerance is thought to be related to glycerol metabolism. However, the regulation of different genes and enzymes under high salt stress conditions has not yet been investigated. In this study, glycerol metabolism was characterized after establishing the optimum salt stress conditions for D. salina Y6. As the salinity in the medium changed from 1 M to 3 M, the glycerol content increased sharply for 1–3 h and then gradually reached its maximum level after 24 h of salt stress (a 2.3-fold increase). Semi-quantitative reverse transcription-polymerase chain reaction analysis showed that the transcription of the glucose-6-phosphate isomerase gene was not affected by salt stress, whereas the transcriptions of the fructose-1, 6-diphosphate aldolase gene, glycerol-3-phosphate dehydrogenase genes-2 and -3, and the dihydroxyacetone reductase gene initially increased and then declined. Furthermore, the magnitude of the decline was greater than that of the increase, particularly for the dihydroxyacetone reductase gene. Enzyme activity analysis showed that the activity of the glucose-6-phosphate isomerase did not change significantly, whereas fructose-1, 6-diphosphate aldolase activity decreased to 63% and dihydroxyacetone reductase activity decreased to 31% after 24 h of salt stress. Therefore, we propose that while the regulation of salt for glycerol metabolism in D. salina is very complex, the reduced decomposition of glycerol helps its accumulation. Finally, we also propose that dihydroxyacetone reductase may be the key enzyme involved in the regulation of the glycerol metabolic pathway under salt stress conditions.