Drying and rewetting (D/W) of soil have significant impacts on soil organic matter (SOM) turnover. We hypothesised that frequent D/W cycles would release the labile organic matter locked away in soil aggregates, increasing the priming effect (PE) (acceleration or retardation of SOM turnover after fresh substrate addition) due to preferential utilisation by microbes. 13C-labelled lignocellulose was added to the soil, and the effects of 0, 1, or 4 cycles of D/W were evaluated at 5 °C and 25 °C after a 27-day incubation of undisturbed soil cores from a temperate forest (Araucaria araucana). Following the incubation, macroaggregates (' 250 μm), microaggregates (250–53 μm), and silt + clay materials (' 53 μm) were separated. For each aggregate size class, three organic matter (OM) fractions (light (fPOM ' 1.6 g cm−3), occluded (oPOM 1.6–2.0 g cm−3), and heavy (Hf ' 2.0 g cm−3) were determined. D/W cycles caused macroaggregates to increase and a decrease in microaggregates (' 15%) at warm temperatures, and preferential use of the novel particulate organic matter (13C labelled), formerly protected fPOM. CO2 efflux was three times higher at 25 °C than at 5 °C. The D/W cycles at 25 °C had a strong negative impact on cumulative CO2 efflux, which decreased by approximately − 30%, induced by a negative PE of −50 mg C kg−1 soil with 1 D/W cycle and − 100 mg C kg−1 soil with 4 D/W cycles, relative to soil under constant soil moisture receiving 13C-labelled lignocellulose, but no cycles. Increasing the temperature and the number of D/W cycles caused a decrease in substrate use efficiency of particulate lignocellulose. In conclusion, D/W cycles at warm temperatures accelerated OM turnover due to preferential use from fPOM, increasing macroaggregates at the expense of microaggregates. A novel pathway of OM release and PE due to the D/W cycles is discussed. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature.