In this paper, direct methanol fuel cell's (DMFC's) electrochemical process was successfully investigated in situ using Electrochemical Impedance Spectroscopy (EIS) method. Under three-electrode system, anode and cathode's polarization overpotential, charge transferring resistance and active surface were independently measured in order to reveal the degradation factors after 50 h stability testing. The results showed that Ru's dispersing, membrane's swelling and water flooding were main reasons resulting in performance decline. And SEM images confirmed these conclusions. Moreover, cathode's degradation was less serious than anode and it could be recovered. Traditional equivalent circuit model of self-breathing DMFC was modified according to a fact that the dielectric relaxation phenomenon of porous electrode occurred at cathode rather than anode. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.