Adding nitrogen substrates to soils can induce short-term changes in soil organic matter (SOM) transformations – a response termed the ‘priming effect’. However, it is unknown how priming effects on nitrous oxide (N2O) emissions can be altered following a strong freeze-thaw cycle. A mesocosm experiment evaluated two soil managements: with and without history of manure applications. These soils were subjected to three moisture regimes: Low, Medium and High. Apart from the controls, which received no N, we banded 15N-labelled urea into these soils representing a typical fall fertilization, and subsequently simulated a wide fall-freeze-thaw cycle, with temperatures from + 2, to -18, and finally + 23°C, respectively. The overall highest N2O production was observed 1 day after thawing. At that time, measurements of N2O site preference indicated that denitrification produced 83% of the N2O flux. Relative to the unamended controls (baseline), adding urea consistently triggered a 24% greater cumulative N2O production specifically originated from SOM following thawing (245 vs. 305 µg N2O-N kg− 1 soil, P = 0.022). This substantiates a positive priming of SOM that manifested shortly after the rapid, wet thawing of the soils. Soils having a manure history or higher moisture also exhibited an augmented production of N2O from SOM (Ps < 0.01). Although the overall priming of SOM was positive, two weeks after thawing, negative priming of daily N2O fluxes also occurred, but only in soils under High moisture. Besides urea additions, the propensity for primed N2O emissions from SOM after thawing was influenced by increasing moisture and earlier manure applications.