The effectiveness of afforestation in sequestrating carbon (C) depends on soil organic C (SOC) stability, yet the underlying mechanisms on the SOC stabilization in response to afforestation remain unresolved. Here, we conducted a random block sampling to explore the changes in SOC relative stability by comparing the proportions of stabilized C in total SOC before and after afforestation. We also calculated microbial C use efficiency (CUE), and analyzed its relation to SOC relative stability to test whether SOC relative stability would be enhanced by increasing microbial CUE following afforestation. The 40 years of afforestation significantly increased the concentration of all C fractions but it decreased SOC relative stability by lowering the proportions of stabilized C in SOC. Microbial CUE showed a significant and positive relationship with the proportions of stabilized C in total SOC. Lower microbial CUE in the afforested soils was primarily associated with lower soil N availability, and higher soil moisture and root density. Additionally, the concentration of stabilized C in the topsoil (0–10 cm) rapidly increased following afforestation and then became level off after total SOC had reached up to 20 g kg⁻¹, while the macroaggregate-occluded C fraction kept fast-growing. Overall, our result revealed the decline in SOC relative stability following afforestation, which was regulated by microbial CUE and the inherent soil C capacity, thereby providing new insights into the assessment of “carbon neutrality” under global afforestation.