Abstract: Microbial residue nitrogen (N) is an important component of soil N pool, with its formation and stabilization playing a crucial role in soil N accumulation. Soil microbial N use efficiency (NUE), i.e., the proportion of total utilized organic N allocated to microbial biomass synthesis, significantly influences microbial residue N accumulation. However, the linkage of soil microbial NUE with microbial N residue remains poorly understood. In this study, soil microbial NUE, microbial N residue, and other soil properties were determined for three mineral soil horizons, i.e., 0~10 cm, 10~20 cm, and 20~40 cm in karst and non-karst forests of Northwest Guangxi. Microbial NUE in the karst forest was 0.41 ± 0.05, 2.93 times higher than that in the non-karst forest (0.14 ± 0.04). Total microbial residue N in the karst forest was 1.32 ± 0.26 g/kg, 2.17 times greater than in non-karst forest (0.61 ± 0.11 g/kg). There was no significant difference in microbial NUE across soil horizons, but total microbial residue N decreased with soil horizon. Microbial NUE was controlled by microbial growth and positively related to the stoichiometric imbalance between soil C:N and microbial biomass C:N. Total microbial residue N was positively related to exchangeable calcium and N-acquisition enzyme activity, but exhibited no significant correlation with microbial NUE. This indicates that exchangeable calcium contributes more dominantly than microbial NUE in promoting microbial residue N accumulation, primarily by protecting it from microbial decomposition through mineral-associated mechanisms. These findings contribute to the understanding of soil microbial N metabolism and N accumulation mechanisms in karst forests.