The unicellular halotolerant green alga species Dunaliella are able to proliferate in extremely varied salinities by synthesizing intracellular glycerol and adjusting the cell shape and volume. However, some marine Dunaliella species such as Dunaliella tertiolecta are not able to regulate cell volume as an immediate response to counter external osmotic shock. Here we report that a rapid shock-response mechanism is present in Dunaliella tertiolecta, involving uptake of exogenous glycerol in response to hyperosmotic shock without changing cell volume, and this glycerol uptake activity is associated with the Dunaliella tertiolecta glycerol uptake protein 1 (DtGUP1) gene, which belongs to the membrane-bound O-acyltransferase. The mutant DtGUP1-E, in which the DtGUP1 gene is silenced, displayed an inability to take up glycerol from the medium and showed cell death under hyperosmotic shock. To our knowledge, this is the first time a gene product has been reported in Dunaliella tertiolecta that is involved in glycerol uptake activity under hyperosmotic stress.