Nitrous oxide reductase (N2OR), the enzyme responsible for the reduction of N2O to N2 in denitrification, uses copper (Cu) as its cofactor. Its activity is lowered under conditions of Cu deficiency. In general, high organic matter (OM) soil decreases Cu availability. The present study investigated different Cu forms, namely organic (ORG) v. inorganic (INO), and associated concentrations (750, 550, 125, 60 μg Cu/g soil) for their efficacy in affecting denitrification and especially N2OR activity in high OM peat soil in a water saturated anaerobic condition for 24 h. Gas and liquid samples were taken every 8 h and analyzed for NO3
−, N2O and N2. Inorganic Cu treatments did not affect N transformation rates and N2OR activity among the different treatments (P > 0.05) throughout the incubation compared with the control (CON). The ORG Cu treatments increased NO3
− (P < 0.05), NO2
− (P < 0.05) and N2O (P < 0.05) transformation rates compared with CON. These changes were ORG Cu dose dependent. N2OR activity increased first in the 750 μg ORG Cu treatment (P < 0.05) during 8 to 16 h followed by the other ORG Cu treatments (P < 0.05) during 16 to 24 h compared with CON. These results highlight the importance of Cu form and concentration on N transformation rate during denitrification. The findings can potentially be applied to systems like soil, wastewater, constructed wetlands, etc., in which reactions of the denitrification pathway are manipulated.