The influence of sulfur deficiency on biomass production was analyzed in the four Chlorellaceae species, Chlorella vulgaris, Chlorella sorokiniana, Chlorella lobophora, and Parachlorella kessleri. Culturing under sulfur-deficient conditions promoted transient accumulation of starch followed by a steady increase in lipid storage. Transmission electron microscopy indicated an increase and decrease in starch granules and subsequent enlargement of lipid droplets under sulfur-deficient conditions. Chlorellaceae spp. accumulated 1.5–2.7-fold higher amounts of starch and 1.5–2.4-fold higher amounts of lipid under sulfur-deficient conditions than under sulfur-sufficient conditions. More than 75% of the fatty acids that accumulated in Chlorellaceae spp. under the sulfur-sufficient condition were unsaturated and culturing under sulfur-deficient conditions increased the saturated fatty acid content from 24.3% to 59.7% only in P. kessleri. These results indicate that the sequential accumulation of starch and lipid is a response to the sulfur depletion that commonly occurs in Chlorellaceae spp.