Natural wetlands are the single largest source of atmospheric methane (CH4). Both a changed climate and deposition of anthropogenic nitrogen and sulfur can alter the production and oxidation of CH4 respectively and thereby also CH4 exchange. We used a long-term (12 years) factorial field experiment in a boreal oligotrophic mire to evaluate the effects of greenhouse cover and addition of ammonium nitrate and sodium sulfate on the production and oxidation of CH4 by applying laboratory incubations of samples from five depths in the mire. The rates of CH4 production were measured without amendments and after the addition of either glucose or sulfate. Twelve years of increased nitrogen deposition has changed the mire from a Sphagnum-dominated plant community to one dominated by sedges and dwarf shrubs. The deposition of nitrogen to the field plots caused increased production of CH4 in incubations without amendments (34%), and also after amendments with glucose (40%) or sulfate (42%). This indicates increased substrate availability (without amendments) but also a greater abundance of methanogens (glucose amendment). The greenhouse cover caused a decrease in CH4 production in incubations without amendments (34%), after glucose amendment (20%) and after sulfate amendment (31%). These responses indicate decreased substrate availability (without amendment) accompanied by the reduced abundance of methanogens (glucose amendment). The field application of sulfur had no effect on CH4 production at the depth where maximal CH4 production occurred. Closer to the mire surface, however, the rate of CH4 production was significantly reduced by 32–45%. These results suggest that the deposition of sulfate has altered the vertical distribution of methanogens and sulfate-reducing bacteria. The oxidation of CH4 was not significantly affected by any of the long-term field treatments.