In this article, an effective method for dephosphorylation of phosphopeptides by cerium oxide is described. The dephosphorylation activity of cerium oxide was evaluated by two standard phosphopeptides and the phosphopeptides in digests of phosphoprotein alpha-casein and beta-casein. Results showed that the dephosphorylation of all the phosphopeptides was completed in 10 min, and temperature had little effect on the dephosphorylation, the dephosphorylation could be carried out at 0 degrees C, room temperature and 37 degrees C. The dephosphorylation mediated by cerium oxide can be attributed to Lewis acid and nucleophile activations. Advantages of using cerium oxide as catalyst for the dephosphorylation include: safe, simple, high catalytic activity, and no precise control of the treatment temperature. The method is valid for the phosphorylation of Ser, Thr and Tyr, and can be used for phosphoprotein analysis.