We investigated the effects of a 5 °C soil + air experimental heating on root and microbial respiration in a boreal black spruce (Picea mariana (Mill.) B.S.P.) forest in northern Manitoba, Canada, that was warmed between 2004 and 2007. In 2007, the 14 C/ 12 C signatures of soil CO 2 efflux and root and soil microbial respiration were used in a two-pool mixing model to estimate their proportional contributions to soil CO 2 efflux and to examine how each changed in response to the warming treatments. In laboratory incubations, we examined whether warming had altered microbial respiration rates or microbial temperature sensitivity. The 14 C/ 12 C signature of soil CO 2 efflux and microbial respiration in the heating treatments were both significantly (p < 0.05) enriched relative to the control treatment, suggesting that C deposited nearer the atmospheric bomb peak in 1963 contributed more to microbial respiration in heated than control treatments. Soil CO 2 efflux was significantly greater in the heated than control treatments, suggesting the acclimation to temperature of either root or microbial respiration was not occurring in 2007. Microbial respiration in laboratory incubations was similar in heated and control soils. This study shows that microbial respiration rates still responded to temperature even after 4 years of warming, highlighting that ecosystem warming can cause a prolonged release of soil organic matter from these soils.