Silicon is a critical element for diatom growth; however our understanding of the molecular mechanisms involved in intracellular silicon responses are limited. In this study, an iTRAQ-LC-MS/MS quantitative proteomic approach was coupled with an established synchrony technique to reveal the global metabolic silicon-response in the model diatom Thalassiosira pseudonana subject to silicon starvation and readdition. Four samples, which corresponded to the time of silicon starvation, girdle band synthesis, valve formation, and right after daughter cell separation (0, 1, 5, 7 h), were collected for the proteomic analysis. The results indicated that a total of 1,831 proteins, representing 16% of the predicted proteins encoded by the T. pseudonana genome, could be identified. Of the identified proteins, 165 were defined as being differentially expressed proteins, and these proteins could be linked to multiple biochemical pathways. In particular, a number of proteins related to silicon transport, cell wall synthesis, and cell-cycle progress could be identified. In addition, other proteins that are potentially involved in amino acid synthesis, protein metabolism, and energy generation may have roles in the cellular response to silicon. Our findings provide a range of valuable information that will be of use for further studies of this important physiological response that is unique to diatoms.