Simple SummaryThe impact of a broiler chicken diet that is supplemented with canola meal (CM) on the cecal microbiota, growth, and metabolome of broiler chickens was examined. CM did not affect feed consumption, weight gain, nor the richness, evenness, or diversity of the cecal bacterial community. However, CM affected the structure of the bacterial microbiota, the concentrations of short-chain fatty acids (SCFAs) were elevated, and the abundance of bacterial taxa known to ferment dietary fiber were more abundant in the ceca of birds fed the CM diet. Nuclear magnetic resonance spectroscopy metabolomics was applied, and a number of metabolites that were associated with SCFA metabolism were differentially regulated in cecal digesta. The supplementation of diet with CM also affected the metabolic profiles of the pancreas, liver, and breast muscle (e.g., metabolites that are associated with energy production, protection against oxidative stress, and pathways of amino acid and glycerophospholipid metabolism). In summary, broiler chickens that were fed a diet supplemented with CM showed equivalent feed consumption and growth, but CM affected the composition and function of the cecal microbiota, and the metabolome of the pancreas, liver, and breast muscle of birds indicating the possibility of an increased disease risk. The study also demonstrated the utility of using metabolomics to ascertain impacts of diet on the microbiota and host tissues, and identified biomarkers to facilitate subsequent evaluation in production settings.The inoculation of one-day-old broiler chicks with the cecal contents from a mature broiler breeder resulted in a highly diverse and uniform cecal bacterial community. CM did not affect feed consumption, weight gain, nor the richness, evenness, or diversity of the cecal bacterial community. However, the structure of the bacterial community was altered in birds fed the CM diet. Although the CM diet was formulated to contain equivalent metabolizable energy to the control diet, it contained more dietary fiber. The abundance of bacterial families, including those that are known to contain species able to metabolize fiber was altered (e.g., bacteria within the families, Methanobacteriaceae, Atopobiaceae, Prevotellaceae, Clostridiales Family XIII, Peptostreptococcaceae, and Succinivibrionaceae), and concentrations of SCFAs were higher in the ceca of birds fed the CM diet. Moreover, concentrations of isoleucine, isobutyrate, glutamate, and 2-oxoglutarate were higher, whereas concentrations of phenyllactic acid, indole, glucose, 3-phenylpropionate, and 2-oxobutyrate were lower in the digesta of chickens that were fed CM. The metabolic profiles of pancreas, liver, and breast muscle tissues of birds fed the CM diet differed from control birds. Metabolites that were associated with energy production, protection against oxidative stress, and pathways of amino acid and glycerophospholipid metabolism had altered concentrations in these tissues. Some of the observed changes in metabolite levels may indicate an increased disease risk in birds fed the CM diet (e.g., pancreatitis), and others suggested that birds mounted metabolic response to offset the adverse impacts of CM (e.g., oxidative stress in the liver).