The human digestive system harbors a vast diversity of commensal bacteria and maintains a symbiotic relationship with them. However, imbalances in the gut microbiota, known as dysbiosis, accompany various diseases, such as inflammatory bowel diseases (IBDs) and colorectal cancers (CRCs), which have a significant impact on the well-being of people globally. Glycosylation of the mucus layer is a key factor that plays a critical role in maintaining the homeostatic environment in the gut. This review delves into the ways in which the gut microbiota, gut epithelial barrier, and immune cells work together to establish a balanced gut environment. Specifically, the role of glycosylation in regulating immune cell responses and mucus metabolism in this process is examined. Additionally, the review explores various modulatory approaches used to maintain or restore the functional gut microbiota.