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Potassium chlorate (KClO₃) has been widely used to evaluate the divergence in nitrogen use efficiency (NUE) between <i>indica</i> and <i>japonica</i> rice subspecies. This study investigated the transcriptional regulation of major genes involved in the NUE in rice treated with KClO₃, which acts as an inhibitor of the reducing activity of nitrate reductase (NR) in higher plants. A set of two KClO₃ sensitive nitrate reductase (NR) and two nitrate transporter (NRT) introgression rice lines (BC2F7), carrying the <i>indica</i> alleles of NR or NRT, derived from a cross between Saeilmi (<i>japonica</i>, P1) and Milyang23 (<i>indica</i>, P2), were exposed to KClO₃ at the seedling stage. The phenotypic responses were recorded 7 days after treatment, and samples for gene expression, physiological, and biochemical analyses were collected at 0 h (control) and 3 h after KClO₃ application. The results revealed that Saeilmi (P1, <i>japonica</i>) and Milyang23 (P2, <i>indica</i>) showed distinctive phenotypic responses. In addition, the expression of <i>OsNR2</i> was differentially regulated between the roots, stem, and leaf tissues, and between introgression lines. When expressed in the roots, <i>OsNR2</i> was downregulated in all introgression lines. However, in the stem and leaves, <i>OsNR2</i> was upregulated in the NR introgression lines, but downregulation in the NRT introgression lines. In the same way, the expression patterns of <i>OsNIA1</i> and <i>OsNIA2</i> in the roots, stem, and leaves indicated a differential transcriptional regulation by KClO₃, with <i>OsNIA2</i> prevailing over <i>OsNIA1</i> in the roots. Under the same conditions, the activity of NR was inhibited in the roots and differentially regulated in the stem and leaf tissues. Furthermore, the transcriptional divergence of <i>OsAMT1.3</i> and <i>OsAMT2.3</i>, <i>OsGLU1</i> and <i>OsGLU2,</i> between NR and NRT, coupled with the NR activity pattern in the roots, would indicate the prevalence of nitrate (NO₃¯) transport over ammonium (NH₄⁺) transport. Moreover, the induction of catalase (CAT) and polyphenol oxidase (PPO) enzyme activities in Saeilmi (P1, KClO₃ resistant), and the decrease in Milyang23 (P2, KClO₃ sensitive), coupled with the malondialdehyde (MDA) content, indicated the extent of the oxidative stress, and the induction of the adaptive response mechanism, tending to maintain a balanced reduction–oxidation state in response to KClO₃. The changes in the chloroplast pigments and proline content propose these compounds as emerging biomarkers for assessing the overall plant health status. These results suggest that the inhibitory potential of KClO₃ on the reduction activity of the nitrate reductase (NR), as well as that of the genes encoding the nitrate and ammonium transporters, and glutamate synthase are tissue-specific, which may differentially affect the transport and assimilation of nitrate or ammonium in rice.