Nitrogen (N) is an important macronutrient for blueberry growth and development. To date, the response mechanism of blueberry to N deficiencystress has not been well elaborated. In this study, the adaptability strategy of blueberries under nitrogen deficiency stress was comprehensively studied using one-year-old seedlings of the highbush blueberry cultivar 'Legacy' subjected to two treatments: normal N fertilization treatment as a control (CK, 15 mM N fertilization) and N deficiency treatment (ND, without N fertilization). The physiological results showed that under nitrogen deficiency stress, plant growth slowed, the dry and fresh weights of the plants decreased, and the chlorophyll (Chl) content decreased significantly. The contents of hydrogen peroxide (H₂O₂), malonaldehyde (MDA), reduced glutathione (GSH), and the activity of superoxide dismutase (SOD) were increased. The absorption of nutrients was significantly affected under nitrogen deficiency treatment. The N, phosphorus (P), potassium (K), sodium (Na) and iron (Fe) contents were decreased, while calcium (Ca), magnesium (Mg), copper (Cu) and zinc (Zn) contents were increased. A total of 1465 differentially expressed genes (DEGs) were obtained in transcriptomic analysis, of which N metabolism-, carbohydrate metabolism- and amino acid metabolism-related pathways were significantly enriched and involved in efficient N uptake and transport. Glycolysis/gluconeogenesis (EMP) and the pentose phosphate pathway (PPP) are the main energy sources in the process of carbohydrate metabolism, and the five upregulated DEGs may play key regulatory roles. We also identified DEGs involved in ions transport, phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, hormone signaling and related transcription factors (TFs). Our results reveal the regulatory mechanisms of the blueberry response to nitrogen deficiency stress and provide new strategies for N nutrient management in blueberry cultivation.