Subsoil (below 20 cm), storing over 50 % of soil organics carbon (SOC) within the 1 m depth, plays a critical role in regulating climate and ecosystem function. However, little was known on the changes in SOC decomposition induced by exogenous C input (i.e., priming effect) across the whole soil profile under nitrogen (N) enrichment and climate warming. We designed an incubation system of soil columns with minor physical disturbance, which allows the manual additions of exogenous C and N and incubation under ambient or elevated temperature. A negative priming effect by glucose was observed in all layers of ambient soil, while the negative priming effect was enhanced by soil depth but inhibited by warming. Nitrogen addition shifted the priming effect from negative to positive under ambient temperature, and decreased the magnitude of negative priming effect under elevated temperature. Nitrogen uplift effect on priming effect was more pronounced in subsoil compared to topsoil, while this effect diminished with rising temperature. Soil microbial activity (e.g., the CO2 production within 3 days) and acid phosphatase activity had important roles in regulating the variations in priming effect across the soil profile. Our results indicated that increase in labile substrate (e.g., exogenous C input) input would not lead to native SOC destabilization in subsoil, N addition shifted the priming effect from negative to positive, increasing the SOC decomposition under ambient temperature, while labile C input together with N addition benefited SOC sequestration by inducing negative priming effects in forest soil under warming climate.