The effects of exercise on nutrient digestion and absorption in the intestinal tract are not well understood. A few studies have reported that exercise training increases the expression of molecules involved in carbohydrate digestion and absorption. Exercise was also shown to increase the blood concentration of glucagon-like peptide-2 (GLP-2), which regulates carbohydrate digestion and absorption in the small intestine. Therefore, we investigated the effects of exercise on the expression of molecules involved in intestinal digestion and absorption, including GLP-2. Six-week-old male mice were divided into a sedentary (SED) and low-intensity exercise (LEx) group. LEx mice were required to run on a treadmill (12.5 m/min, 1 h), whereas SED mice rested. All mice were euthanized 1 h after exercise or rest, and plasma, jejunum, ileum, and colon samples were collected, followed by analysis via IHC, EIA, and immunoblotting. The levels of plasma GLP-2 and the jejunum expression of the GLP-2 receptor, sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) were higher in LEx mice. Thus, we showed that acute low-intensity exercise affects the expression of molecules involved in intestinal carbohydrate digestion and absorption via GLP-2. Our results suggest that exercise might be beneficial for small intestine function in individuals with intestinal frailty.