The objective of this study was to investigate the effect of different phosphorus concentrations on growth, biomass, photosynthetic pigment, and lipid content of green microalgae Chlorella pyrenoidosa under nitrogen deficiency. Four types of media including control with sufficient nitrogen and phosphorus (240 mg/L for N, 5.56 mg/L for P) and three various phosphorus concentrations (5.56, 27.8, and 55.6 mg/L) under limited nitrogen (8 mg/L) were applied under photoautotrophic culture for 5 days. The findings demonstrated that high phosphorus concentration had a marginal impact on the growth and biomass concentration. Moreover, the combination of phosphorus repletion and nitrogen deficiency had an unfavorable effect on Chl a+b and carotenoid. It is interesting to note that lipid content was considerably accumulated by a slight excess of phosphorus (27.8 mg/L) with nitrogen deficiency. This was 53% higher than that of control and 12% higher than that of sufficient phosphorus with nitrogen deficiency. This study indicates that increasing phosphorus concentration up to 27.8 mg/L under nitrogen deficiency is an effective strategy to enhance the lipid content of C. pyrenoidosa in autotrophic culture.