AimsSoil nitrogen (N) availability is crucial for ecosystem structure and function. However, in the degraded karst areas of southwest China, the mechanisms governing soil N availability and microbial community contributions during natural restoration remain unclear.MethodsThis study conducted plant surveys, soil analyses and metagenomic sequencing across three restoration stages of a degraded karst forest (TG: shrubland, SG: secondary-growth forest, OG: old-growth forest) in Guizhou Province, China, to explore changes in soil microbial nitrification and its impact on soil N dynamics.ResultsNatural restoration promoted the synchronous recovery of plant communities and soil nutrients. Soil N availability (NO3-/NH4+ ratio) increased from 6.48 in TG to 44.26 in OG, driven by higher NO3- levels linked to enhanced microbial nitrification. The expansion of the overall nitrifying microbial community was mainly due to improved soil nutrients, which increased soil N availability. Among the nitrification processes, ammonia oxidation (AO) and nitrite oxidation (NO) increased with soil N availability, whereas hydroxylamine oxidation (HO) was decreased. Structural equation modeling (SEM) further suggested that AO was the primary driver of increased N availability during restoration. The large differences in microbial composition among the three nitrifying communities highlight significant functional differentiation in microbial roles within N cycling.ConclusionsNatural restoration of degraded karst forests enhances soil N availability primarily through promoting microbial nitrification, especially the AO process, driven by soil nutrient levels. This study provides new insights into microbial-mediated N cycling and offers valuable guidance for the ecological restoration of karst ecosystems.