Scenedesmus sp. 14-3 was identified as a suitable candidate for producing biodiesel. The present work studied the effects of nitrogen concentration on the biomass and lipid productivity of algae, the consumption of sodium nitrate, and the two-stage N-deficient cultivation that could enhance dramatically the accumulation of biomass and lipids of Scenedesmus sp. 14-3. The two-stage N-deficient cultivation was described as follows: microalga Scenedesmus sp. 14-3 was cultured under low light intensity (LL) for 10 days in an N-deficient medium by 20 % inoculum concentration, and transferred to complete N-depletion BG11 under high light intensity (HL) for 8 days. The highest lipid content of Scenedesmus sp. 14-3 was 53.05 ± 0.08 % (10 % inoculum concentration) following the second stage of N-deficient cultivation after 8 days. For the second stage of N-deficient cultivation, the lipid content of Scenedesmus sp. 14-3 was 49.85 ± 0.22 %, which was 1.8 times higher than that under low light intensity (LL) (46–48 μmol m−2 s−1 ) in 10 days. Meanwhile, the high algal biomass productivity was around 0.10 g L−1 day−1 after the first stage of N-deficient cultivation (10 days) and the biomass productivity was around 0.037 g L−1 day−1 under the second stage of N-deficient cultivation (8 days). The comparison under different culture conditions showed a significant effect of the two-stage of N-deficient cultivation on lipid accumulation of Scenedesmus sp. 14-3. The two-stage N-deficient cultivation without centrifugation achieved a complete N-depletion condition, but the two-stage process required centrifugation which is unsuitable for commercialization and large-scale utilization. In summary, two-stage N-deficient cultivation is a more suitable and effective culture method for commercial applications and dramatic accumulation of lipids than the two-stage process.