Nitrogen (N) is essential for the growth of algae, and its concentration varies greatly in the ocean, which has been regarded as a limitation for phytoplankton growth. Despite its great importance, most of the existing studies on the mechanisms underlying the effects of N on diatoms have focused on physiology, biochemistry and a few target genes and have rarely involved whole genomic analyses. Therefore, in this study, we integrated physiological data with RNA and protein profiling data to reveal the response strategy of Thalassiosira pseudonana under N-depleted conditions. Physiological measurements indicated that the cell growth capacity and chlorophyll content of the cells decreased, as did the expression of photosynthesis- and chlorophyll biosynthesis-related genes or proteins. The RNA-Seq profile results showed that T. pseudonana responded to N deprivation through increases in glycolysis, the TCA cycle and N metabolism as well as down-regulation in the Calvin cycle, gluconeogenesis, pentose phosphate, oxidative phosphorylation and lipid synthesis. These results provide a basic understanding for further research addressing how N affects phytoplankton in terms of genomics.