Nitrogen (N) and phosphorus (P) limitations induce neutral lipid accumulation and membrane lipid remodeling in the domesticated oleaginous microalga Tisochrysis lutea. This study compared growth, photosynthetic activity, biochemical and transcriptional responses of T. lutea throughout batch cultures under N or P limitation compared with non-limiting nutrient condition (NP). The results show that, under N limitation, the breakdown and re-use of cellular N-containing compounds contributed to carbohydrates and further triacylglycerol (TAG) accumulation, where the accumulation of docosahexaenoic acid (DHA) in neutral lipids might mainly be derived from the recycling of membrane polar lipids. Conversely, P limitation did not hinder the photosynthetic capacity; a higher efficiency of carbon fixation fueled the allocation of carbon fluxes to the reserves of carbohydrates and neutral lipids. These latter accumulated without massive degradation of essential cellular compounds. Betaine lipids constitute the major compounds of non-plastidial membranes in T. lutea. Given an extremely low constitutive phospholipid level, phospholipids might not be involved in the regulation of P storage. However, transfer of P from non-plastidial to chloroplast membranes might occur, keeping a stable lipid composition of thylakoid membranes and maintaining a relatively high photosynthetic capacity under P deprivation.