Coccolithophores are phytoplankton widely distributed in the global oceans and play a key role in biological carbon fixation. Particulate organic carbon (POC) is produced through photosynthetic carbon fixation, and particulate inorganic carbon (PIC) is produced during calcification and coccoliths formation. In addition, the dense coccoliths can increase the sinking rate of POC, and accelerate the output of POC driven by biological pumps to the deep ocean. In this study, we investigated the physiological and molecular responses of cells under different nitrogen concentrations using two strains of coccolithophores, Emiliania huxleyi (BOF-92 and RCC1266). It was found that the pigment abundance of the two strains decreased, but the inorganic carbon content of the cells increased significantly under nitrogen limitation. For the RCC1266, which had higher levels of calcification, nitrogen limitation also increased cellular organic carbon content. Combined with transcriptome data, the molecular mechanism of the physiological response was explored. We found that the C4 pathway, with phosphoenolpyruvate carboxylase (PEPC) as the key, complements the weakened Calvin cycle to maintain photosynthesis. Meanwhile, enhanced upregulation of PEPC, formamidase (FMD) and GS/GOGAT genes maintained nitrogen metabolism. The upregulation of Ca^{2 +} and inorganic carbon related transporter genes offer possibilities in enhanced calcification. This study provides us with a fundamental understanding of changes in cellular carbon content and molecular mechanisms of coccolithophores under nitrogen limitation. It is an important basis for exploring how marine nutrient limitation affects the efficiency of phytoplankton biological pumps in the future.