The oxidation of graphene layer on Ru(0001) has been investigated by means of scanning tunneling microscopy. Graphene overlayer can be formed by decomposing ethyne on Ru(0001) at a temperature of about 1000 K. The lattice mismatch between the graphene overlayer and the substrate causes a moire pattern with a superstructure in a periodicity of about 30 angstrom. The oxidation of graphene/Ru(0001) was performed by exposure the sample to O-2 gas at 823 K. The results showed that, at the initial stage, the oxygen intercalation between the graphene and the Ru(0001) substrate takes place at step edges, and extends on the lower steps. The oxygen intercalation decouples the graphene layer from the Ru(0001) substrate. More oxygen intercalation yields wrinkled bumps on the graphene surface. The oxidation of graphene, or the removal of carbon atoms can be attributed to a process of the combination of the carbon atoms with atomic oxygen to form volatile reaction products. Finally, the Ru(0001)-(2 x 1)O phase was observed after the graphene layer is fully removed by oxidation. (C) 2010 Elsevier B.V. All rights reserved.