Abstract: As the primary source of water for drinking and irrigation, groundwater necessitates analysis of the key controlling factors governing uranium contamination, thereby providing theoretical support for the management and control of groundwater pollution. Nitrate ( \mathrmN\mathrmO_3^- ), a prevalent anthropogenic contaminant of groundwater and a potent oxidant, can facilitate the activation and migration of uranium through complex interactions involving chemical constituents and microbial activity. However, the optimal conditions governing \mathrmN\mathrmO_3^- -induced U activation under the combined influence of multiple factors remain poorly defined. A deeper understanding of this \mathrmN\mathrmO_3^- -mediated process and its controlling factors is essential to elucidate the stability and oxidative release mechanisms of uranium in groundwater systems. To address this critical knowledge gap, the present study employed a comprehensive suite of laboratory-based static batch experiments, utilizing authentic groundwater as the reaction matrix. This study systematically investigated the effects of \mathrmN\mathrmO_3^- concentration, Geobacter metallireducens abundance, pH, and Fe²⁺ concentration on the rate of \mathrmN\mathrmO_3^- -mediated U activation. It quantified the influence of \mathrmN\mathrmO_3^- on U oxidation rates across these variables, thereby clarifying its regulatory role and underlying mechanisms in U activation and migration. Results indicate that groundwater pH exhibits a triphasic pattern: an initial increase (acid consumption), followed by a decrease (acid production), and a final increase (acid consumption). Optimal U activation occurrs at pH 6.8 and 7.0. Variations in Eh are primarily driven by changes in Fe²⁺ and U(Ⅵ) concentrations. The production of Fe³⁺ and U(Ⅵ) during activation significantly increases system Eh. Under the experimental conditions, higher Eh values correspond to more effective U activation. The U activation rate shows a positive correlation with initial \mathrmN\mathrmO_3^- concentration, stimulating both Geobacter metallireducens growth and U activation. Above 100 mg/L \mathrmN\mathrmO_3^- , the activation rate plateau, although activation continues. Within the optimal growth pH range for Geobacter metallireducens (6.8-7.0), both bacterial activity and U activation rates are enhanced. Deviation from this pH range inhibits bacterial respiration, consequently reducing U activation. The \mathrmN\mathrmO_3^- -mediated U activation rate increases with higher initial Fe²⁺ concentrations, promoting Geobacter metallireducens activity and U activation. Beyond 0.2 mg/L Fe²⁺, further concentration increases yielded diminishing returns in activation rate. Collectively, these results provide crucial mechanistic insights into the biogeochemical factors governing \mathrmN\mathrmO_3^- -mediated U mobilization in groundwater. The identification of optimal pH conditions, concentration thresholds for key reactants ( \mathrmN\mathrmO_3^- , Fe²⁺), and the pivotal role of specific microbial metabolism (Geobacter metallireducens) significantly advances predictive capability regarding U contamination dynamics. This foundational understanding is vital for developing effective strategies to forecast, prevent, and remediate uranium pollution in vulnerable aquifer systems, ultimately safeguarding water resources.