When dry soil is re-wet there is a rapid increase in CO2 efflux and rates can remain above those of well-watered controls for one or more days. These large pulses of CO2 efflux are known as the “Birch effect”. To provide experimental evidence of different pools of C fuelling the Birch effect, we incubated a drying soil with 13C6-glucose, re wet the soil and quantified 13C labelling of pools (microbial biomass, trehalose, extracellular, and old C) and soil CO2 efflux. We took advantage of trehalose being the most 13C-enriched pool (δ13C = +518‰) to obtain direct isotopic evidence of trehalose's contribution to respiration and microbial growth. For soil incubated with 13C6-glucose, the δ13C of soil respiration was +35‰ in dry soil, increased to 100‰ in the 10 min following rewetting, and subsequently decreased. During the first 5 h after re-wetting, trehalose must have been contributing to respiration given that δ13C of soil respiration was more 13C enriched than trehalose-free microbial biomass (δ13C = +30‰), extracellular C (δ13C = −17.7‰), and old C (δ13C = −22.9‰). A four-member isotopic mixing model suggested trehalose underpinned 16% of respiration in the 1st hour after rewetting, decreasing to 7% in the fifth hour. At times beyond 5 h after rewetting, trehalose underpinned 0–4% of respiration. In the seven days following rewetting microbial biomass increased 2292 nmol C g−1. Isotopic mass balance indicated trehalose-C could account for no more than 5% of the gross influx of C into microbial biomass, instead the increase in microbial biomass was fuelled by un-enriched or weakly labelled pools such as old C and extracellular C. Collectively these data provide direct experimental evidence C from trehalose does not significantly contribute to microbial growth in re-wet soil, but instead contributes to respiration for the first 5 h after rewetting.