In this report, we performed a comparative study of NxCNTs at three different N doping levels as the anode and cathode catalyst support in both rotating disk electrode (RDE) and realistic direct methanol fuel cell (DMFC) test. Our results show that nitrogen doping can play significantly conflicting roles in the anode and cathode performance: (i) A very low doping level (1at.%) is sufficient to induce a homogenous distribution of both anode (PtRu) and cathode (Pt) catalyst nanoparticles. (ii) In RDE test, while nitrogen doping can substantially increase the specific activity of the anodic methanol oxidation, it brings no specific activity enhancement in the cathodic oxygen reduction reaction (ORR), suggesting that nitrogen doping can hardly affect the ORR electrocatalysis on the Pt surface. (iii) In MEA test, a low nitrogen doping level at 1 at.% in NxCNTs as anode catalyst support resulted in a higher power density compared to non-doped CNT support at both 30 and 60°; increasing nitrogen-doping content decreased the power density, likely due to a decreased electron conduction. In contrast, at the cathode, NxCNTs support mainly showed a lower power density, particularly at 60°, which is rationalized by the increased hydrophicility and thus unflavored removal of the water. Taken together, our results provide important guidelines for application of NxCNT support at the appropriate electrode of DMFCs.