© 2015 Springer International Publishing Switzerland The decomposition of soil organic carbon (SOC) is intrinsically sensitive to temperature. However, the degree to which the temperature sensitivity of SOC decomposition (as often measured in Q10 value) varies with soil depth and labile substrate availability remain unclear. This study explores (1) how the Q10 of SOC decomposition changes with increasing soil depth, and (2) how increasing labile substrate availability affects the Q10 at different soil depths. We measured soil CO2 production at four temperatures (6, 14, 22 and 30 °C) using an infrared CO2 analyzer. Treatments included four soil depths (0–20, 20–40, 40–60 and 60–80 cm), four sites (farm, redwood forest, ungrazed and grazed grassland), and two levels of labile substrate availability (ambient and saturated by adding glucose solution). We found that Q10 values at ambient substrate availability decreased with increasing soil depth, from 2.0–2.4 in 0–20 cm to 1.5–1.8 in 60–80 cm. Moreover, saturated labile substrate availability led to higher Q10 in most soil layers, and the increase in Q10 due to labile substrate addition was larger in subsurface soils (20–80 cm) than in surface soils (0–20 cm). Further analysis showed that microbial biomass carbon (MBC) and SOC best explained the variation in Q10 at ambient substrate availability across ecosystems and depths (R2 = 0.37, P < 0.001), and MBC best explained the variation in the change of Q10 between control and glucose addition treatment (R2 = 0.14, P = 0.003). Overall, these results indicate that labile substrate limitation of the temperature sensitivity of SOC decomposition, as previously shown in surface soils, is even stronger for subsoils. Understanding processes controlling the labile substrate availability (e.g., with rising atmospheric CO2 concentration and land use change) should advance our prediction of the fate of subsoil SOC in a warmer world.