The effect of sulfur limitation on the partitioning of carbon, nitrogen, and sulfur was investigated in Dunaliella salina. D. salina was able to adapt to 6 μm sulfate; under these conditions, the cells showed reduced growth and photosynthetic rates. Whereas intracellular sulfate was depleted, phosphate, nitrate, and ammonium increased. Amino acids showed a general increase, and alanine became the most abundant amino acid. The activities of four key enzymes of carbon, sulfur, and nitrogen metabolism were differentially regulated: Adenosine 5′ triphosphate sulfurylase activity increased 4-fold, nitrate reductase and phosphoenolpyruvate (PEP) carboxylase activities decreased 4- and 11-fold, respectively, whereas carbonic anhydrase activity remained unchanged. Sulfur limitation elicited specific increase or decrease of the abundance of several proteins, such us Rubisco, PEP carboxylase, and a light harvesting complex protein. The accumulation of potentially toxic ammonium indicates an insufficient availability of carbon skeletons. Sulfur deficiency thus induces an imbalance between carbon and nitrogen. The dramatic reduction in PEP carboxylase activity suggests that carbon was diverted away from anaplerosis and possibly channeled into C3 metabolism. These results indicate that it is the coordination of key steps and components of carbon, nitrogen, and sulfur metabolism that allows D. salina to adapt to prolonged sulfur limitation.