Tidal flats are zones of high carbon burial yet also hotspots of organic matter (OM) mineralization, where episodic inputs of labile carbon can reshape the fate of indigenous OM through priming effects. However, the temporal dynamics of mineralization and the microbial mechanisms remain poorly constrained. Here, we incubated intertidal sediments from the Nanhui shoal tidal flat (East China Sea) with ¹³C-glucose for 72 h and quantified response across multiple scales by combining interfacial oxygen and CO₂ flux measurements, compound-specific ¹³C analysis of phospholipid fatty acids (PLFAs), and metagenomic sequencing. Glucose addition triggered a rapid increase in diffusive oxygen uptake (DOU), peaking after ~12 h at 28–29 mmol m⁻² d⁻¹ (2–3 times fold higher than the control) before returned to baseline after 48 h, while CO₂ efflux exhibited a similar temporal pattern. During the later incubation phase, the apparent respiratory quotient (ARQ) in the mid-intertidal zone increased from ~0.9 to ~2.4, coinciding with near-complete depletion of overlying-water NO₃⁻, indicating an emerging anaerobic contribution to respiration, likely driven by nitrate reduction. ¹³C enrichment was strongly concentrated in the 0–1 cm surface layer and declined to natural abundance below 2 cm, with the highest labelling in the Gram-negative bacterial markers C16:1ω7c and C18:1ω7c (reaching 29–47 at%). Metagenomics analyses further revealed extreme enrichment of opportunistic heterotrophic degraders, particularly Psychromonas in the surface layer (up to ~25-fold), together with significant enrichment of carbohydrate-metabolism and glycoside-hydrolase genes (GH13, GH3, GH1), whereas overall community structure was governed primarily by depth rather than by substrate addition. This study provides provides mechanistic evidence linking microbial carbon assimilation, community succession, and sediment respiration, offering new insights into how tidal-flat carbon cycling responds to episodic inputs of labile organic matter.