Fens, which extend over vast areas in the Northern hemisphere, are sources of the greenhouse gas CH4. Climate change scenarios predict a lowering water table (WT) in mires. To study the effect of WT drawdown on CH4 dynamics in a fen ecosystem, we took advantage of a WT drawdown gradient near a ground water extraction plant. Methane fluxes and CH4 production and oxidation potentials were related to microbial communities responsible for the processes in four mire locations (wet, semiwet, semidry, and dry). Principal component analyses performed on the vegetation, pH, CH4, and WT results clearly separated the four sampling locations in the gradient. Long-term lowering of WT was associated with decreased coverage of Sphagnum and aerenchymatic plants, decreased CH4 field emissions and CH4 production potential. Based on mcrA terminal restriction fragment length polymorphism the methanogen community structure correlated best with the methane production and coverage of aerenchymatic plants along the gradient. Methanosarcinaceae and Methanocellales were found at the pristine wet end of the gradient, whereas the Fen cluster characterized the dry end. The methane-oxidizing bacterial community consisted exclusively of Methylocystis bacteria, but interestingly of five different alleles (T, S, R, M, and O) of the particulate methane monooxygenase marker gene pmoA. The M allele was dominant in the wet locations, and the occurrence of alleles O, S, and T increased with drainage. The occurrence of the R allele that characterized the upper peat layer correlated with CH4 oxidation potential. These results advance our understanding of mire dynamics after long-term WT drawdown and of the microbiological bases of methane emissions from mires.