The increase in atmospheric nitrous oxide (N2O), a potent greenhouse and ozone depleting gas, is of serious global concern. Soils are large contributors to this increase through microbial processes that are enhanced in agricultural land due to nitrogenous fertilizer applications. Denitrification, a respiratory process using nitrogen oxides as electron acceptors in the absence of oxygen, is the main source of N2O. The end product of denitrification is benign dinitrogen (N2) and understanding what regulates the shift in ratio of N2O and N2 emission is crucial for mitigation strategies. The role of organic carbon in controlling N2O reduction is poorly understood, and mostly based on application of glucose. Here we investigated how a range of carbon compounds (succinate, butyrate, malic acid, acetate, glucose, sucrose and cysteine) affect denitrifier N2/N2O production stoichiometry under laboratory conditions. The results show that a soil's capability in efficiently reducing N2O to N2 is C substrate dependent and most compounds tested were different in regards to this efficiency compared to glucose. We challenge the concept of using glucose as a model soil C compound in furthering our understanding of denitrification and specifically the efficiency in the N2O reductase enzyme. Organic acids, commonly exuded by roots, increased N2/N2O ratios compared to glucose, and therefore mitigated net N2O release and we suggest provides better insights into soil regulatory aspects of N2O reduction. The widespread use of glucose in soil laboratory studies could lead to misleading knowledge on the functioning of denitrification in soils with regards to N2O reduction.