CO2 release from forest soils (Rs) is a prominent flux in the global carbon cycle. Rs is derived from roots (autotrophic respiration, Ra) and microbial (heterotrophic) respiration and is highly dynamic, as it depends on edaphic and environmental conditions as well as root functional traits and microbial community composition. It is unclear how root functional traits affect root and microbial respiration rates; however, their consideration may help parse out the relative contributions of root and microbial respiration to Rs. At a temperate forest site, root systems of 3–4 functional root orders and their surrounding surface soil were carefully excavated and placed into custom trays designed to repeatedly measure Rs in situ on eight temperate tree species that varied in their root functional strategies and mycorrhizal affinity. Rs was measured bi-weekly to monthly for nearly one year using a custom chamber attached to a gas exchange system. Rs varied over time, ranging from 0.3 to 12 µmol m−2 s−1. Comparable root systems of the same species were excised from the soil and specific root respiration rates (Rr) were measured. Rr ranged from 2.5 to 9.0 nmol g−1 s−1 and was negatively correlated with root tissue density and positively related to root tissue nitrogen concentration. Using Rr to estimate Ra, we estimate that Ra accounts for <10%, on average 2–3%, of Rs for individual root systems (averaging 1.2 g dry biomass) housed in surrounding soil (average 1.3 kg dry mass) in situ; thus, Ra was roughly 20 times greater than Rh per unit mass. The contribution of Ra peaked in the fall and coincided with leaf senescence of the forest canopy. A soil-sterilizing experimental treatment designed to help isolate Ra in situ reduced bacterial biomass and shifted fungal community composition, but there was no reduction in Rs of the in-situ root-soil tray systems. The relative Ra to Rs ratio increased with root functional strategies characterized by greater specific root length and tip abundance, but also to greater root tissue density. The ratio of Ra to Rs also increased with warmer soil temperatures and decreased slightly with increasing soil moisture. We discuss how incorporating root functional traits as modulators of the autotrophic contribution to Rs could be considered when modeling total soil CO2 efflux from forests.