Very-low-calorie diets (VLCD) have been used to obtain rapid weight loss. This study examined the effect of prolonged dieting on brush border disaccharidases and the potential of glutamine supplementation to alter this response. Female Osborne-Mendel rats were made obese on a high-fat diet and then randomly assigned to either a control (C) group or to 1 of 3 weight loss groups. Using a very-low-calorie diet (VLCD) which represented a 75% reduction in kcal intake, a low-calorie diet (LCD) which was a 50% reduction in kcals, or a VLCD supplemented with glutamine (VLCD+GLN), the rats were reduced to 250–260 g. The proximal small intestine was removed to assay disaccharidase and alkaline phosphatase activity and mucosal contents of DNA, RNA, and protein. Compared to C, VLCD animals showed significantly increased lactase and sucrase activity. Maltase activity was significantly higher in VLCD than all other groups. No significance differences were found among the groups for mucosal DNA, RNA, protein or alkaline phosphatase activity. The VLCD diet demonstrated support and increased activity in brush border enzymes. Glutamine supplementation of the VLCD neither prevented a decrease in proximal mucosal weight nor benefited disaccharidase activity.