ABSTRACT 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 . IMPORTANCE Previous studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum -dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process, remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy and that members of the Alphaproteobacteria ( Rhizobiales ) catalyze this process at the bog surface using the molybdenum-based form of the nitrogenase enzyme.