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Untargeted metabolomics has been increasingly used to evaluate metabolic alterations caused by diet, disease, or other factors in animals. The purpose of this exploratory study was to evaluate the impact of Cannabidiol (CBD) supplementation on the canine carboxyl and hydroxyl submetabolomes. Sixteen dogs (18.2 ± 3.4 kg BW) were utilized in a completely randomized design with treatments consisting of control and 4 mg CBD/kg BW/d. After 21 d of treatment, blood was collected approximately 2 h after morning treat consumption. Plasma collected from samples was analyzed using CIL/LC-MS-based untargeted metabolomics to analyze carboxyl- and hydroxyl-containing metabolites. Metabolites that differed (fold change (FC) ≥ 1.2 or ≤ 0.83 and FDR ≤ 0.05) between the two treatments were identified using a volcano plot. Biomarker analysis based on Receiver Operating Characteristic (ROC) curves was performed to identify biomarker candidates (area under ROC ≥ 0.90) of the effects of CBD supplementation. Volcano plot analysis revealed that 42 carboxyl-containing metabolites and 32 hydroxyl-containing metabolites were differentially altered (FC ≥ 1.2 or ≤ 0.83, FDR ≤ 0.05) by CBD; these metabolites were involved in the metabolism of lipids, amino acids, carbohydrates, and more. Biomarker analysis identified 23 carboxyl-containing metabolites and 15 hydroxyl-containing metabolites as candidate biomarkers of the effects of CBD (area under ROC ≥ 0.90; P