Aeromonas salmonicida, an important pathogenic bacterium which induces furunculosis, is globally causing increased risks in Atlantic salmon (Salmo salar) farming. Although the kidney is the main target organ of A. salmonicida, the metabolic profiling of kidney in response to A. salmonicida in vivo remains unknown. Here, we used 1H nuclear magnetic resonance (NMR) to comprehensively analyze the metabolic changes in the kidney of Atlantic salmon. Through the NOESYPR1D spectrum combined with multi-variate pattern recognition analysis, including principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) models, significant metabolic changes were observed seven and 14 days post-infection and in a control group. Hence, the main objective of this study was to estimate the significant metabolites with resistance to furunculosis and further understand the mechanism of A. salmonicida in Atlantic salmon. Notably, substantial alterations of kidney metabolites were observed, such as with fumarate, alanine, valine, glycine, aspartate, choline, glycerophosphocholine and betaine, and summarized by metabolic pathways including the citrate cycle, glycolysis/gluconeogenesis, tryptophan metabolism, and urea cycle, respectively. Changes were also observed in 3-hydroxybutyrate and phosphocholine which were not involved in these four metabolic pathways. After analyzing the alteration trend of these metabolites, we inferred that A. salmonicida caused absorption inhibition of amino acids and disturbed protein metabolism as well as cell metabolism in favor of its replication. These observations offered novel insights into the mechanisms of infection at a functional level and facilitated further assessment and clarification of fish disease from A. salmonicida exposure.