Silvopastoral systems that integrate trees in pasture production systems are likely to enhance soil carbon (C) storage in lower soil layers due to the presence of deep tree roots. To quantify the relative soil C contribution from trees (C3 plants) and warm season grasses (C4 plants) in silvopastoral systems, soil samples were collected and analyzed from silvopastures of slash pine (Pinus elliottii)+bahiagrass (Paspalum notatum), and adjacent open pasture (OP), at six depths down to 125 cm, at four sites representing two major soil orders (Spodosols and Ultisols) of Florida. The plant sources of C in whole (nonfractionated) and three soil fraction sizes (250–2000, 53–250, and <53 μm) were traced using stable C isotope signatures. The silvopasture sites contained higher amounts of C3-derived soil organic carbon (SOC) compared with OP sites, at all soil depths. Slash pine trees (C3 plants) seemed to have contributed more C in the silt+clay-sized (<53 μm) fractions than bahiagrass (C4 plants), particularly deeper in the soil profile. Spodosols sites contained more C in the <53 μm fraction at and below the spodic horizon (occurring between 15 and 50 cm) in silvopasture compared with OP. The results indicate that most of SOC in deeper soil profiles and the relatively stable <53 μm C fraction were derived from tree components (C3 plants) in all the sites, suggesting that the tree-based pasture system has greater potential to store more stable C in the soil compared with the treeless system.