Chronic ethanol consumption is associated with not only the alteration of metabolic profiles in biofluids but also the composition of the gut microbiome. Our understanding of the importance of the intestinal microbiota as well as the disturbances elicited by ethanol intervention is limited by the fact that previous analyses have primarily focused on biofluids and liver tissue metabolome; the metabolic profiles of the gastrointestinal (GI) contents are rarely investigated. In this study, we applied a metabonomics approach using a high performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF MS) and gas chromatography-mass spectrometry (GC-MS) to characterize the metabolic alterations of the contents within the GI tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) in male Sprague-Dawley rats following 8 weeks of ethanol exposure. We obtained a snapshot of the distinct changes of the intestinal content metabolite composition in rats with ethanol exposure, which indicated a profound impact of ethanol consumption on the intestinal metabolome. Many metabolic pathways that are critical for host physiology were affected, including markedly altered bile acids, increased fatty acids and steroids, decreased carnitines and metabolites involved in lipid metabolism, a significant decrease of all amino acids and branched chain amino acids, and significantly decreased short chain fatty acids except for acetic acid, which rapidly elevated as a product of ethanol metabolism. These results provide an improved understanding of the systemic alteration of intestinal content metabolites in mammals and the interplay between the host and its complex resident microbiota and may aid in the design of new therapeutic strategies that target these interactions.