While excessive nitrogen fertilizer application enhances crop yields, it does so at the expense of ecosystem health, making the enhancement of nitrogen use efficiency (NUE) an imperative for sustainable agriculture. This study investigates the mechanisms underlying the response to low nitrate (LN) stress in two Brassica juncea genotypes: a high-NUE (H158) and a low-NUE (L159), aiming to identify genetic resources for improved NUE. Hydroponic experiments revealed that H158 displayed enhanced antioxidant capacity, nitrogen assimilation, and nitrogen uptake efficiency (NUpE) under LN conditions relative to L159. Field trials corroborated these findings, with H158 demonstrating higher yields and greater agronomic nitrogen use efficiency (ANUE) across a gradient of nitrogen application rates. Genomic analysis identified 1,654 genes associated with NUE, including NIN-LIKE PROTEIN 4 (BjuB04.NLP4), whose G/C variants influenced transcriptional regulation and root-to-shoot ratio responses to nitrogen availability. Notably, BjuB04.NLP4-Hap2 (H158) was predominant in low-nitrogen soils, while BjuB04.NLP4-Hap1 (L159) was more common in high-nitrogen soils. Collectively, our findings uncover and characterize valuable genetic resources for breeding rapeseed varieties with enhanced NUE, providing both elite germplasm and functional molecular markers.