Emiliania huxleyi is a marine phytoplankton that plays a critical role in global carbon and sulfur cycling. The genome of E. huxleyi has been sequenced, and an in-depth proteomic profile of this organism has been reported. This study analyzed the phosphoproteome of E. huxleyi and identified its changes under calcium-limited conditions. A TiO 2 microcolumn was used for phosphopeptide enrichment, followed by liquid chromatography-tandem mass spectrometry analysis. Overall, we identified 7,010 phosphorylated sites on 3,355 phosphopeptides associated with 2,929 phosphoproteins in E. huxleyi. Quantitative analysis revealed changes in the phosphoproteome in E. huxleyi when ambient conditions changed to calcium-limited conditions, notably the phosphorylation of some transporters was altered. This study provides an overview of protein phosphorylation in E. huxleyi and paves the way for further investigations of its biological functions.