Abstract Background and aims Biological nitrification inhibition (BNI) is a chemical ecological phenomenon whereby plants specifically suppress nitrification by releasing inhibiting compounds from roots, an effective strategy for improving nitrogen uptake by limiting nitrogen losses from agricultural fields. During this study, we have aimed at characterizing hydrophilic BNI activity released from maize roots to understand the chemical basis for BNI function in maize. Methods Maize plants were grown hydroponically and root exudates were collected using water-based solutions and hydrophilic BNI activity was extracted. We isolated the target BNI compounds by a combination of chromatographic techniques and bioassays using a recombinant luminescent ammonia-oxidizing bacterium Nitrosomonas europaea (pHLUX20). Results We identified 6-methoxy-2(3H)-benzoxazolone (MBOA) as the responsible BNI compound with a median effective dose (ED₅₀) = 0.76 μM. MBOA inhibited the conversion of NH₃ to NH₂OH as well as NH₂OH to NO₂⁻ in N. europaea, suggesting that MBOA blocks both ammonia monooxygenase and hydroxylamine oxidoreductase enzymatic pathways. Treatment with MBOA significantly suppressed NO₂⁻ and NO₃⁻ production during soil incubation, but this activity was reduced subsequently due to biodegradation of MBOA by soil microbes. A quantification experiment revealed that MBOA accounted for nearly 50% of the total BNI activity in hydrophilic and hydrophobic exudates from maize roots. A soil incubation test showed that two previously identified benzoxazinoids, HDMBOA and HDMBOA-β-glucoside, can be eventually transformed into MBOA. Conclusion We elucidated MBOA as the key component of BNI in maize. Collectively, the present findings will serve as the groundwork for construction of an advanced environment-friendly agricultural system.