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Nitrous oxide (N2O) is a potent greenhouse gas with high global warming potential. Soil acts as sources and sinks of N2O. The mechanism of soil N2O uptake is important for both the net emission of N2O and the N2O concentration in the atmosphere. The use of organic matter as an amendment is an effective management practice for soil fertility worldwide, whereas, its effects on soil N2O uptake remain unclear. To understand the responses of N2O uptake with organic carbon incorporation. A laboratory incubation using 15N2O dilution tracing technology with and without acetylene (C2H2) was conducted. The result showed that gross rate of soil N2O production in Glucose treatment (3.33 g kg−1 glucose incorporated with soil) with and without C2H2 were 497.9 and 639.2 μg N kg−1 soil h−1, which were significantly higher than that in CK (no carbon incorporation) and Straw (3.33 g kg−1 straw incorporated with soil) treatments (25.3 and 29.8 μg N kg−1 soil h−1 and 42.0 and 52.5 μg N kg−1 soil h−1, respectively). The gross rates of N2O uptake was significantly increased by Glucose with 221.2 μg N kg−1 soil h−1, whereas, in CK and Straw treatments, they were 21.6 and 28.3 μg N kg−1 soil h−1. However, the gross rates of N2O uptake were decreased to 2.2, 3.2 and 6.2 μg N kg−1 soil h−1 by C2H2 for CK, Straw and Glucose treatments, which indicated that denitrification could be a key pathway of soil N2O uptake. We also found that soil dissolved organic carbon (DOC) content was an important factor that positively correlated with the gross rates of N2O production and uptake. These results indicate that soil DOC enhances both soil N2O production and uptake, and denitrification is an important pathway of soil N2O uptake.