An experiment was conducted to determine effects of betaine on growth performance and intestinal health in rabbits fed diets with different levels of digestible energy. During a 36-day experiment, a total of 144 healthy 35-day-old weaned New Zealand white rabbits with a similar initial body weight (771.05 ± 41.79 g) were randomly distributed to a 2 × 3 factorial arrangement. Experimental treatments consisted of two levels of digestible energy (normal: 10.20, and low: 9.60 MJ/kg) and three levels of betaine (0, 500, and 1000 mg/kg). Results indicated that rabbits fed the diet with low digestible energy (LDE) had reduced body gain/feed intake (G:F) on days 1-14 and 1-36 (P < 0.05), increased the apparent total tract digestibility (ATTD) of neutral detergent fiber (NDF), acid detergent fiber (ADF) and n-free extract, and decreased the ATTD of gross energy (GE), crude fiber (CF) and organic matter (P < 0.05). The LDE diet up-regulated the gene abundance levels of duodenum junctional adhesion molecule-3 (JAM-3), and down-regulated the ileum toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and interleukin-6 (IL-6) (P < 0.05). Activities of amylase, lipase, trypsin, and the immunoglobulin M content in the jejunum were decreased in LDE treatment group (P < 0.05). Dietary betaine supplementation increased the ATTD of GE, dry matter (DM), ADF and n-free extract by LDE (P < 0.05). The villus height, crypt depth and goblet cell numbers were decreased, and the villus crypt ratio was increased in the duodenum (P < 0.05). The gene abundance levels of duodenum interleukin-2 (IL-2) were down-regulated, and the duodenum junctional adhesion molecule-2 (JAM-2) and junctional adhesion molecule-3 (JAM-3) were up-regulated (P < 0.05). Furthermore, addition of betaine to the LDE diet increased the ATTD of GE, DM and organic matter in rabbits (P < 0.05). Gene abundance level of ileum IL-6 and duodenum JAM-3 were up-regulated (P < 0.05). In summary, LDE diets can reduce the activity of intestinal digestive enzymes and decrease the ATTD of nutrients. However, the addition of betaine to LDE diets improved the intestinal barrier structure and nutrient ATTD in rabbits, with better results when betaine was added at an additive level of 500 mg/kg.