The continuing increase in fuel demand, the dramatic situation of climate changes and global warming are bringing worldwide attention to identification of alternative energy source that can replace fossil fuel. Among all the feedstock considered for biodiesel production, microalgae are the most promising. In particular, it has been demonstrated that microalgae enhance their neutral lipids content in stress conditions, such as nutrient starvation. However, this increase in neutral lipids content is often coupled with a strong biomass decrease. In this study, in order to limit this side effect, a novel three-phase growth strategy was tested on the diatom Skeletonema marinoi. After a first phase directed to biomass production the culture, during a second phase of semi-continuous regimen characterized with reduction of nitrate concentration, achieved a neutral lipids content per cell of 10% of dry weight with slight impact on biomass. After these two phases the cells, gradually acclimatized to low nitrogen concentration, were still able to grow in a third maturation phase performed in batch. During this last phase, the culture further increased neutral lipid content up to the 30 % of dry weight.