Chlorella strains have great potential as a resource for production of biofuels due to their relatively fast growth rate and easy cultivation. Heterotrophic cultivation of Chlorella has been developed due to higher biomass concentration and accumulation of much higher lipid content than classical photoautotrophic culture. Instead of using glucose, using low cost materials, such as starch or cellulose-hydrolyzed solution, is a good strategy to reduce the cost of culture medium and the cost of microalgae-based biofuels from heterotrophic fermentation of Chlorella. Potato starch is relatively cheap material in comparison with cornstarch or cassava starch, hence it could be an ideal carbon source for cultivating Chlorella. In a number of oil-producing microalgal species, Chlorella has the capacity of accumulating much higher proportion of fatty acids by nitrogen starvation. The objective of this study was to investigate the effects of various nitrogen resources on cell growth and lipid accumulation in heterotrophic Chlorella vulgaris using potato starch hydrolysate (PSH) as the sole carbon source. In order to illustrate the influence of various nitrogen sources on biomass and lipid content in C. vulgaris, the effects of these nitrogen source including inorganic and organic nitrogen source such as alanine or tyrosine on cell growth and lipid accumulation of C. vulgaris were examined. As the sole carbon source PSH was added to SE medium for algal cultivation prior to addition of all nitrogen sources. NaNO3 and alanine were added to yield starting concentrations of 1.5 mmol /L, 3.0 mmol/L and 6.0 mmol/L, respectively. Tyrosine was added to yield starting concentrations of 0.75 mmol/L, 1.5 mmol /L and 3.0 mmol/L, respectively. Continuous cultivation of C. vulgaris was carried out under dark condition for 10 days. The cell number, special growth rate, cellular chlorophyll content, neutral lipid content and total lipid content of C. vulgaris under present cultural condition were determined throughout the entire experimental period. The results showed that: (1) Higher chlorophyll content and biomass production were observed with higher nitrate-nitrogen concentration, although low concentrations of nitrate-nitrogen can achieve high lipid accumulation in C. vulgaris. The highest biomass productivity was obtained at 1.5 mmol/L sodium nitrate, the biomass of 2.65 g/L, lipid concentration of 51.21% and the total lipid production of 1.36 g/L were achieved. (2) When alanine was used as the sole nitrogen source, it was also observed that the cell growth and total chlorophyll content increased but the lipid content reduced. (3) A low level of biomass production was observed while adding tyrosine as a nitrogen source; however, cell enlargement and high chlorophyll content were achieved under this condition, resulting in high lipid content of 38.78%-47.02%. Based on above results, it was concluded that the unicellular alga C. vulgaris was able to respond to nitrogen nutrient changes by inducing specific amino acid transport systems, and it seemed to have developed more specific mechanisms of amino-acid uptake and lipid production along with subtle regulatory phenomena such as induction by nitrogen starvation. Furthermore, tyrosine, as the part of the third uptake system induced by glucose transport system, can also enhance continually cellular lipid accumulation in C. vulgaris.