Background: Agricultural intensification induces prolonged impacts on soil acidification (SA), whereas linking the mechanisms therein to structural and functional evolution of soil microbiota remain unclear, especially over longer time scales. Methods: To investigate the short- and long-term effects of agricultural intensification on soil microbiome and its association with SA severity, we performed metagenomic sequencing of soil samples from typical rice-vegetable rotations for 0 (control, V0), 10 (V10), 20 (V20) years. The objectives were to clarify biomarkers that indicative of SA and its putative functions of acid production. Results: Rice-vegetable rotations for 0, 10, 20 years yielded three well-defined soil clusters with differential acid saturation (V0: 4.5%; V10: 11.8%; V20: 40.7%). Soil pH declined significantly (p < 0.05) from the 6.38 (V0) to 4.82 (V20). Acid cations (H + , Al 3+ ) dominating V20’s exchangeable cation pool, suggesting that soils in V20 were highly acidic and acid-sensitive, which recruited distinct microbiomes. The increased acid cations and NO 3 - -N concentrations are driving forces that lead to a higher abundance of Rhodanobacter , Gemmatirosa , Sphingomonas and Streptomyces in V20 samples, which acting as aciduric biomarkers that significantly and positively correlated with soil acidity. Furthermore, functional modules of microbial genes revealed V20 samples contained more genes associated with“oxidative phosphorylation” and “two-component system”, particularly enriched multiple pathways of cytoplasmic pH homeostasis. More specifically, we found increased genetic abundances of active efflux of protons and cytoplasmic proton consumption in V20 samples over than in V0, which are both involved in the microbial dehydrogenation and acid production, and favoured the growth of Rhodanbacter , Gemmatirosa , Sphingomonas , Streptomyces . Conclusions: Collectively, the identified aciduric biomarkers was closely associated with SA severity in intensive agroecosystems. An overall increase with the ongoing of intensive cropping history in the abundance of indicative genes associated with dehydrogenation and acid production pathways. The differential community compositions provide new clues to elucidate the interaction between the soil microbiome and SA severity, and varying dehydrogenation and acid production pathways provide novel insights into the exacerbation of SA. Keywords: Agricultural intensification, Soil acidification, Metagenomics, Soil microbiome, pH homeostasis