Nitrogen (N) is a vital nutrient for crop growth and development which influences both yield and quality. Melatonin (MT), a known enhancer of abiotic stress tolerance, has been extensively studies; however, its relationship with nutrient stress, particularly N, and the underlying regulatory mechanisms of MT on N uptake remain unclear. In this study, exogenous MT treatment was found to improve the tolerance of apple plants to N deficiency. Apple plants overexpressing the MT biosynthetic gene N-acetylserotonin methyltransferase 9 ( MdASMT9) was used to further investigated the effects of endogenous MT on low-N stress. The overexpression of MdASMT9 improved the light harvesting and heat transfer capability of apple plants, thereby mitigating the detrimental effects of N deficiency on the photosynthetic system. Proteomic and physiological data analyses indicated that MdASMT9 overexpression enhanced the trichloroacetic acid (TCA) cycle and positively modulated amino acid metabolism to counteract N-deficiency stress. Additionally, both exogenous and endogenous MT promoted the transcription of MdHY5, which in turn bound to the MdNRT2.1 and MdNRT2.4 promoters and activated their expression. Notably, MT-mediated promotion of MdNRT2.1 and MdNRT2.4 expression in an MdHY5-dependent manner, ultimately enhancing N absorption. Taken together, these results may provide useful insights into the relationship between MdASMT9-mediated MT biosynthesis and N uptake under N-deficiency conditions in apple plants.