The dysbiosis of intestinal microbiota and their metabolites is linked to the occurrence and development of metabolic syndrome. Although fructose has been proven to be associated with worsened mucus in the colon, its mechanism remains unclear. In this study, we evaluated the relatively low intake of sucrose and fructose in the experimental colitis of Sprague Dawley rats by investigating the microbiome and metabolome. Results showed that sucrose and fructose significantly reduced body weight, colon length and increased inflammation infiltration in colon. Sucrose and fructose worsen colon functions by inhibiting the expression of tight junction (TJ) protein ZO-1 and increasing the level of lipopolysaccharide neoandrographolide (LPS) in plasma, while fructose was more significant. Furthermore, sucrose and fructose significantly changed the composition of gut microbiota characterized by decreasing Adlercreutzia, Leuconostoc, Lactococcus and Oscillospira and increasing Allobaculum and Holdemania along with reducing histidine, phenylalanine, arginine, glycine, aspartic acid, serine, methionine valine, alanine, lysine, isoleucine, leucine, threonine, tryptophan, tyrosine, proline, citrulline, 4-hydroxyproline and gamma amino butyric acid (GABA). Metabolome results showed that fructose may aggravate experimental colitis symptoms by inducing amino metabolism dysbiosis in the colon. These findings suggested that fructose worsened colitis by manipulating the crosstalk between gut microbiota and their metabolites.