This study investigates the metabolic disruptions caused by nicotine (NIC) exposure, with a particular focus on amino acid and lipid metabolism, and evaluates resveratrol (RSV) as a potential protective agent. Mice were divided into four groups: control (CON), NIC-exposed, NIC + RSV-treated, and RSV-only. NIC exposure resulted in significant weight loss, elevated glucose levels, altered lipid profiles, and organ damage, particularly in the liver and kidneys. Increased inflammation was evidenced by elevated levels of IL-6 and CRP. In contrast, RSV treatment mitigated these effects by improving lipid profiles, glycemic indices, and reducing inflammatory markers. Histopathological analysis confirmed reduced tissue damage in the NIC + RSV group compared to the NIC-alone group. Metabolomics analysis using LC-MS/MS revealed significant dysregulation in lipid, amino acid, and nucleotide metabolism in NIC-exposed mice. Fold-change analysis identified altered metabolites, including sphingomyelin 36:1;02 (p < 0.001), valine (p < 0.001), triacylglycerol 4:0-18:1 (p < 0.001), and ceramide 32:1;02 (p < 0.001). Amino acids such as arginine, phenylalanine, glutamic acid, tyrosine, and lysine, as well as NIC metabolites like nornicotine and cotinine, were identified, underscoring molecular fragmentation analysis findings. RSV treatment partially restored metabolic balance, highlighting its role as a metabolic modulator. This study underscores the therapeutic potential of RSV in alleviating NIC-induced metabolic dysfunctions by restoring lipid homeostasis and reducing inflammation. Additionally, it emphasizes the importance of RSV in addressing NIC-related metabolic impairments and the need for noninvasive biomarkers for early disease detection.