There is a knowledge gap on biochar carbon (C) longevity and its priming effects on soil organic carbon (SOC) and recent root-derived C under field conditions. This knowledge would allow the potential of biochar in long-term soil C sequestration to be established. However, most studies on biochar C longevity and its priming effect have been undertaken in plant-free laboratory incubations.A 388 d field study was carried out in the presence of an annual ryegrass (C3) growing on a rhodic ferralsol with established C3/C4 plant-derived SOC (δ13C: −20.2‰) in a subtropical climate. A 13C-depleted hardwood biochar (δ13C: −35.7‰, produced at 450 °C) was applied at 0 and 30 dry t ha−1 and mixed into the top 100-mm soil profile (equivalent to 3% w/w). We report on the differentiation and quantification of root respiration and mineralisation of soil-C and biochar-C in the field. Periodic 13CO2 pulse labelling was applied to enrich δ13C of root respiration during two separate winter campaigns (δ13C: 151.5–184.6‰) and one summer campaign (δ13C: 19.8–31.5‰). Combined soil plus root respiration was separated from leaf respiration using a novel in-field respiration collar. A two-pool isotope mixing model was applied to partition three C sources (i.e. root, biochar and soil). Three scenarios were used to assess the sensitivity associated with the C source partitioning in the planted systems: 1) extreme positive priming of recent SOC derived from the current ryegrass (C3) pasture; 2) equivalent magnitude of priming of SOC and labile root C; and 3) extreme positive priming of the native C4-dominant SOC.We showed that biochar induced a significant negative priming of SOC in the presence of growing plants but no net priming was observed in the unplanted soil. We also demonstrated the importance of experimental timeframe in capturing the transient nature of biochar-induced priming, from positive (day 0–62) to negative (day 62–388). The presence/absence of plants had no impact on biochar-C mineralisation in this ferralsol during the measurement period. Based on a two-pool exponential model, the mean residence time (MRT) of biochar varied from 351 to 449 years in the intensive pasture system to 415–484 years in the unplanted soils.