Pokkah boeng disease (PBD) is a foliar disease causing severe losses in sugarcane crop production. Research into resistance mechanisms against the causal agent, Fusarium verticillioides, is particularly important for farmers and researchers. This work based on the comprehensive analysis of metabolic, proteomic, and bioinformatics data on nitrogen (N) metabolism, which revealed that this biosynthetic reactions was closely related to resistance mechanisms in the sugarcane– F. verticillioides interaction. Our results suggested that pathogen infection reduced the suppression of nitrate reductase (NR) activity, reducing ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) assimilation, which reduces glutamine synthetase (GS), glutamate synthetase (GOGAT) activity and polynucleotide synthesis and promotes RNA degradation, resulting in a decrease in ribosome levels and protein synthesis. Cysteine was found to be associated with the symptoms of PBD, while alanine, lysine, proline, and glutamic acid were found to be involved in protective and regulatory mechanisms as well. Additionally, glutamate played an important role in sugarcane defense against pathogens through the biosynthesis of proline and polyamines. Cyanamide, glutamate, proline, tyrosine, and arachidonic acid metabolism actively participate in resistance and response to stress. C5XPZ6 and C5XCA6 were considered to be critical proteins and key effectors according to this study. In conclusion, we have identified potential proteins and pathways involved in sugarcane resistance to F. verticillioides, revealing new findings that may be useful in the design of future diagnostics or sugarcane protection strategies and providing new insights into the molecular mechanisms of sugarcane–pathogen interactions.