Even a small net increase in soil organic carbon (SOC) mineralization will cause a substantial increase in the atmospheric CO2 concentration. It is widely recognized that the SOC mineralization within deep critical zones (2 to 12 m depth) is slower and much less influenced by anthropogenic disturbance when compared to that of surface soil. Here, we showed that 20 years of nitrogen (N) fertilization enriched a deep critical zone with nitrate, almost doubling the SOC mineralization rate. This result was supported by corresponding increases in the expressions of functional genes typical of recalcitrant SOC degradation and enzyme activities. The CO2 released and the SOC had a similar 14C age (6000 to 10,000 years before the present). Our results indicate that N fertilization of crops may enhance CO2 emissions from deep critical zones to the atmosphere through a previously disregarded mechanism. This provides another reason for markedly improving N management in fertilized agricultural soils.