Microbial N 2 fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO 2 and are susceptible to the changing climate. The objectives of this study were (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in an ombrotrophic Sphagnum -dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O 2 , CO 2 , and CH 4 ) on potential rates of diazotrophy measured by acetylene (C 2 H 2 ) reduction and 15 N 2 incorporation. A molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria ( Bradyrhizobiaceae and Beijerinckiaceae ). Despite higher concentrations of dissolved vanadium ([V] 11 nM) than molybdenum ([Mo] 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing, and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water contents. Incorporation of 15 N 2 was suppressed 90% by O 2 and 55% by C 2 H 2 and was unaffected by CH 4 and CO 2 amendments. These results suggest that peatland diazotrophy is mediated by a combination of C 2 H 2 -sensitive and C 2 H 2 -insensitive microbes that are more active at low concentrations of O 2 and show similar activity at high and low concentrations of CH 4 .