| Summary: | Agriculture is the main contributor to nitrous oxide (N<sub>2</sub>O) emission, but the emission intensity can be controlled by various factors, in particular, the activity of earthworms, one of the most common groups of soil invertebrates. We conducted an incubation experiment to evaluate N<sub>2</sub>O emission in earthworm soil samples compared to non-earthworm ones with applications of high (200 kg ha<sup>−1</sup>) and low (50 kg ha<sup>−1</sup>) mineral N fertilizer doses. We assessed the cumulative N<sub>2</sub>O emission, the dynamics of the soil dissolved organic carbon, and the soil microbial carbon and nitrogen content, as well as the number of <i>nirK</i> and <i>nirS</i> gene copies in bulk soil samples and in isolates from the earthworms’ gut. Our study showed a significant role of the earthworm activity in changing the intensity of N<sub>2</sub>O emission after the application of mineral N fertilizers. The main factor leading to an increase in nitrous oxide emission in the presence of earthworms is the stimulation of free-living soil denitrifiers by the organic matter of the earthworms’ excretions, as well as the thorough mixing of plant residues and soil. Contrary to our expectations, earthworms did not increase the representation of nitrite reductase genes in soil, although the earthworm’s gut can be considered as a refugium for denitrifiers. Our results indicate a possible risk of increased N<sub>2</sub>O emission from arable temperate soils with an increase in earthworm populations as the climate warms, even if application rates of mineral fertilizers are reduced.
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