【Objective】Fluctuation in water levels has a significant influence on the bioavailability of zinc (Zn) and its ecotoxicological impact. However, the effects of water regimes on Zn isotope fractionation and gene expression, and the subsequent role of water management modulation on Zn uptake and transport in rice remains timidly explored. Thus, this study elucidates the mechanisms controlling Zn mobilization and translocation in a soil-rice system at the jointing stage under two contrasting water management regimes (flooding and drainage).【Method】A controlled pot experiment was conducted to investigate Zn isotope fractionation in soil pools, pore water, and rice organs. Zn stable isotopes were measured across bulk soil, HCl- and CaCl2-extractable fractions, iron plaques on root surfaces, and pore water. Rice plants were sampled for roots, stems, leaves, and shoots, and gene expression analyses of key Zn transporters were performed using quantitative real-time PCR. The genes included OsZIP3, OsZIP4, OsZIP5, OsZIP7a, OsZIP8, OsZIP9, OsHMA2, OsHMA3, OsNAS3, OsNAAT1, OsTOM2, and OsYSL15. Isotope fractionation values (Δ66Zn) were calculated between different soil and plant pools, and water management effects were assessed.【Result】 Zn isotope fractionation in soil exhibited similar patterns under both water regimes. Relative to bulk soil, HCl-extractable and CaCl2-extractable Zn and root-surface iron plaques were enriched in heavier Zn isotopes, whereas pore water was enriched in lighter Zn isotopes. The mean fractionation values were Δ66ZnHCl?soil = 0.40‰ ± 0.05‰, Δ66ZnCaCl2?soil = 0.45‰ ± 0.03‰, and Δ66Znpore water?soil = ?0.095‰ ± 0.035‰. Zn uptake by rice plants displayed water-regime-dependent isotope fractionation. Specifically, under flooding, Δ66Znrice?pore water = 0.16‰ ± 0.09‰, whereas under drainage, Δ66Znrice?pore water = 0.06‰ ± 0.08‰. In contrast, Zn translocation from roots to shoots (Δ66Znshoot?root = 0.32‰ to 0.36‰) and from stems to leaves (Δ66Znleaf?stem = ?0.11‰ to ?0.09‰) was largely unaffected by water regime, suggesting that translocation fractionation is less sensitive to water management. At the molecular level, flooding significantly upregulated root expression of OsZIP5 and OsZIP9, responsible for Zn uptake, and OsNAAT1, OsTOM2, and OsYSL15, which mediate the synthesis and secretion of deoxymugineic acid (DMA) and uptake of Zn-DMA complexes. Flooding also increased OsHMA3 expression in roots, facilitating vacuolar sequestration of Zn, while concurrently suppressing OsHMA2 expression, which mediates root-to-shoot transport of Zn2+. Meanwhile, the expression of root-to-shoot transporters OsZIP3, OsZIP4, OsZIP7a, and OsZIP8, as well as OsNAS3 involved in nicotianamine synthesis, was modulated to maintain Zn homeostasis and promote xylem loading. These coordinated gene expression patterns indicate that flooding enhances root absorption of Zn(II)-DMA and heavier Zn2+ through specific ZIP transporters and facilitates the translocation of both Zn(II)-nicotianamine (NA) complexes and Zn2+ to shoots via OsYSL15 and other dedicated transporters.【Conclusion】The integration of Zn stable isotope fractionation with gene expression data reveals that water management strongly influences Zn uptake and transport mechanisms in rice. Flooding promotes the preferential uptake of heavier Zn isotopes via DMA chelation and ZIP-mediated pathways, enhances vacuolar Zn sequestration, and coordinates efficient root-to-shoot translocation, whereas drainage reduces isotopic fractionation during uptake but maintains root-to-shoot transport. This study provides mechanistic insights into the water-regime-dependent regulation of Zn absorption and translocation, highlighting the role of specific transporters and chelators in controlling Zn isotope fractionation and its movement through the soil-rice system. These findings offer a foundational understanding for optimizing Zn nutrition in rice under different water management strategies and can inform agronomic interventions aimed at improving micronutrient use efficiency.