Phosphorus (P) is one of the key nutrients for the growth of phytoplankton. In this study, we used a method coupling label-free quantitation with liquid chromatography-mass spectrometry (LFQ-LC-MS/MS) to track the change of relative protein abundance between P-replete and P-deficient treatments in a non-model dia-tom, Thalassiosira weissflogii. Out of the 631 proteins identified, 132 were found to have significant changes in abundance (>1.5 folds) between the two treatments, especially those proteins involved in macromolecu-lar biosynthesis pathways. For example, the up-regulation of sulfolipid biosynthesis protein in the P-defi-cient culture suggested a switch from using phospholipids to sulfolipids. In addition, the ribosome subunits and tRNA synthetases were down-regulated, which might explain the decrease in protein content in the P-deficient culture. A vacuolar sorting receptor homologous protein was found to be 9.2-folds up-regulated under P-deficiency, indicating an enhancement in the vacuolar sorting pathway for protein degradation. Our results show that T. weissflogii has sophisticated responses in multiple macromolecular metabolism pathways under P-deficiency, a mechanism which can be critical for this species to survive under various levels of P availability in the environment.