Cold stress is a critical abiotic stress that reduces crop yield and quality.The response of the rice proteome to cold stress has been documented, and differential proteomic analysis has provided valuable information on the mechanisms by which rice adapts to cold stress.A global analysis of the change in protein phosphorylation status in response to cold stress remains to be explored, however.Here, we performed a phosphoproteomic analysis of rice roots following exposure to cold stress using a two-dimensional gel electrophoresis-based multiplex proteomic approach.Differentially expressed proteins and phosphoproteins were detected and identified by matrix-assisted laser desorption ionization time of flight/time offlight mass spectrometry combined with querying rice protein databases.Nineteen protein gel spots (stained with silver) showed a twofold difference in abundance of protein spots from gels with and without cold stress;these proteins were identified to be involved in redox homeostasis, signal transduction, and metabolism.Twelve of the thirteen phosphoprotein gel spots (stained with Pro-Q Diamond) that showed a twofold abundance difference were identified, including the following nine proteins: enolase, glyceraldehyde-3-phosphate dehydrogenase, nucleoside diphosphate kinase, ascorbate peroxidase, adenosine kinase, CPK1 adapter protein 2, ATP synthase subunit alpha, methionine synthase 1, and tubulin.Phosphorylation site predictors were used to confirm that the identified proteins had putative phosphorylation sites.These results suggest that phosphorylation of some proteins in rice roots is regulated in response to cold stress.