The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis, but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga , and were shown to be induced by nutrient starvation and/or salt stress. Heterologous expression of GPD2, a putative chloroplastic GPDH, and GPD3, a putative cytosolic GPDH, in a yeast Δ mutant demonstrated the functionality of both enzymes. knockdown mutants for and showed no difference in growth but displayed significant reduction in TAG concentration compared with the wild type in response to phosphorus or nitrogen starvation. Overexpression of and in gave distinct phenotypes. overexpression lines showed only subtle metabolic phenotypes and no significant alteration in growth. In contrast, overexpression lines displayed significantly inhibited growth and chlorophyll concentration, reduced glycerol concentration, and changes to lipid composition compared with the wild type, including increased abundance of phosphatidic acids but reduced abundance of diglycerides, triglycerides, and phosphatidylglycerol lipids. This may indicate a block in the downstream glycerolipid metabolism pathway in overexpression lines. Thus, lipid engineering by GPDH modification may depend on the activities of other downstream enzyme steps. These results also suggest that GPD2 and GPD3 GPDH isoforms are important for nutrient starvation-induced TAG accumulation but have distinct metabolic functions.