Microbial nitrogen (N) demand substantially influences soil N transformations during vegetation succession, its dynamics across contrasting successional pathways remain poorly understood yet. We used vector model, GeoChip 5.0, and 15N-tracer to investigate microbial N metabolism and gross N transformation across primary succession (20–130 years post-glacier retreat) and secondary succession (grassland to primary forest) in the eastern Tibetan Plateau. During primary succession, microbial N limitation was progressively alleviated, coincident with increasing plant biomass and richness, as well as the accumulation of soil labile carbon (C), total N, and phosphorus (P). Increases in soil C, N, and P pools enhanced both gross and net N mineralization rates directly and indirectly by elevating ureC abundance and 4-β-N-acetylglucosaminidase activity. Microbial N limitation emerged in the late coniferous stage of secondary succession. This pattern was associated with declines in plant richness, reductions in soil labile C and pH from early stage (grassland/shrubland) or mid-successional broadleaf stages to late coniferous stages, and concomitant decreases in leucine aminopeptidase activity. Such changes led to a lower gross N mineralization rate and reduced soil N availability in late secondary forests. Overall, our results indicate that microbial N limitation is gradually relieved during soil development following glacier retreat as plant communities and soil C and nutrients become more favorable, whereas late-stage secondary coniferous forests experience microbial N limitation driven by more homogeneous vegetation and reduced soil N mineralization rates. These findings imply that succession-specific management strategies are needed to conserve soil N cycling and ecosystem resilience in fragile subalpine ecosystems.