Haptophytes are a diverse monophyletic group with a worldwide distribution, known to be significantly involved in global climate regulation in their role as a carbon sink. Because nitrogen is a major limiting macronutrient for phytoplankton in oceans and for cultures of microalgae, understanding the involvement of nitrogen availability in haptophyte carbon partitioning is of global and biotechnological importance. Here, we made an ecophysiological study coupled with comprehensive large scale proteomic analysis to examine differences of behavior in reaction to nitrogen availability changes between a wild type strain of Tisochrysis lutea (WTc1) and a mutant strain (2Xc1) known to accumulate more storage lipids. Strains were grown in chemostats and studied under different ecophysiological conditions including N limitation, N repletion and N depletion. Whereas short time N repletion triggered consumption of carbohydrates in both strains, storage lipid degradation and accumulation during changes of ecophysiological status were recorded in 2Xc1 but not in WTc1. After 3 months of continuous culture, 2Xc1 exhibited an unexpected increase of carbon sequestration ability (+ 50%) by producing twofold more carbohydrates for the same nitrogen availability. Deep proteomic analysis by LC-MS/MS identified and compared the abundance of 4332 proteins, i.e. the deepest coverage of a microalgal proteome obtained to date. Results revealed that storage lipid accumulation is favored by an overall reorganization of carbon partitioning in 2Xc1 cells that increases the metabolism of carbon and energy acquisition, and decreases mitochondrial activity and metabolic conversion of storage lipids to phosphoenolpyruvate before gluconeogenesis.