In order to study the effect of phosphorus on biodiesel production from Scenedesmus obliquus especially under nitrogen deficiency conditions, six types of media with combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion were investigated in this study. It was found that nitrogen starvation compared to nitrogen repletion enhanced biodiesel productivity. Moreover, biodiesel productivity was further strengthened by varying the supply level of phosphorus from depletion, limitation, through to repletion. The maximum FAMEs productivity of 24.2 mg/L/day was obtained in nitrogen depletion with phosphorus repletion, which was two times higher than that in nutrient complete medium. More phosphorus was accumulated in cells under the nitrogen starvation with sufficient phosphorus condition, but no polyphosphate was formed. This study indicated that nitrogen starvation plus sufficient P supply might be the real "lipid trigger". Furthermore, results of the current study suggest a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production.