Cattle production is a large source of greenhouse gas (GHG) emissions from the Canadian livestock sector. Efforts to reduce CH4 emissions from enteric fermentation have led to modifications of diet composition for livestock, resulting in a corresponding change in manure properties. We studied the effect of applying manure from cattle fed a barley-based diet with and without the methane inhibitor supplement, 3-nitrooxypropanol (3-NOP), on soil GHG emissions. Three soils common to Alberta, Canada, were used: a Black Chernozem, a Dark Brown Chernozem, and a Gray Luvisol. We compared the supplemented (3-NOPM) and non-supplemented manure (BM) amendments to a composted 3-NOPM (3-NOPC) amendment and a control with no manure amendment (CK). In an 84-day laboratory incubation experiment, 3-NOPM had significantly lower cumulative CO2 emissions compared to BM in both the Black Chernozem and Gray Luvisol. The cumulative N2O emissions were lowest for 3-NOPC and CK and highest for 3-NOPM across all soil types. Cumulative CH4 emissions were only affected by soil type, with a net positive flux from the fine-textured Gray Luvisol and Dark Brown Chernozem and a net negative flux from the coarse-textured Black Chernozem. Cumulative anthropogenic GHG emissions (CO2-equivalent) from soil amended with 3-NOPM were significantly higher than those for both BM and CK amendments in the Black Chernozem, while the cumulative anthropogenic GHG emissions from the 3-NOPC treatment were similar to or significantly lower than those for the BM and CK treatments across all soil types. We conclude that soil GHG emissions resulting from the 3-NOPM amendment are dependent on soil type and 3-NOPM could potentially increase soil GHG emissions compared to BM or CK. Although we show that the composting of 3-NOPM prior to soil application can reduce soil GHG emissions, the composting process also releases GHGs, which should also be considered in assessing the life-cycle of manure application. Our results provide a first look at the potential effect of the next stage in the life cycle of 3-NOP on GHG emissions. Further research related to the effect of soil properties, particularly in field studies, is needed to assess the best management practices related to the use of manure from cattle-fed diets supplemented with 3-NOP as a soil amendment.