A convenient method of investigating the degradation of a direct methanol fuel cell (DMFC) was carried out at highly anodic potential (i.e., 0.8 V). Accelerating degradation of a DMFC was investigated by electrochemical methods, X-ray diffraction (XRD), transmission electron microscopy (TEM), electron probe microanalysis (EPMA), and X-ray photoelectron spectroscopy (XPS). The degradation was preliminarily diagnosed and predicted with electrochemical impedance spectroscopy and verified with microscopic examination. The EPMA and XPS results showed that the sulfonic acid vanished in the broken anodic layer compared to the original one, which results in an increase of internal resistance (Rs). The increase of interfacial (Rif) and electrochemical reaction resistance (Rrxn) in the degraded cell might be a result from catalytic degradation. Ru dissolves from the anodic catalyst to decrease the catalytic activity then reduced near the cathode. The evidence for Ru dissolution was determined by CO stripping combined with EPMA and XPS analyses. Its reduction near the cathode was observed through TEM and EPMA. The catalysts in both the anode and cathode aggregated to decrease the catalytic activity in the degradation process by XRD and TEM. On the basis of the electrochemical study and related analysis, we proposed a mechanism for this accelerating degradation of DMFC.