In order to investigate the effects of dietary supplementation with Jerusalem artichoke (Helianthus tuberosus L.; JA) as prebiotic, 72 Wistar rats were allocated into 4 groups (JA-0, JA-2, JA-4 and JA-6) and fed for 12 weeks a basal diet supplemented with 0, 2, 4 and 6% of pulverized JA tubers. The JA addition did not affect the growth performance of animals and had no detrimental effect on feed intake, however fibre digestibility was improved (linear, P = 0.003) whereas dry matter and organic matter digestibility (linear, P = 0.012 and 0.001, respectively) were decreased. Apparent digestibility of calcium and phosphorus was increased (quadratic, P = 0.002 and 0.005, respectively) in rats fed diets with JA supplementation. The JA addition into diets significantly (P ≤ 0.05) increased the populations of beneficial Lactobacillus spp. and Bifidobacterium spp. microbiota in the caecal, colonic and rectal digesta. Acetic acid (linear, P = 0.019), propionic acid (linear, P = 0.008) and total short-chain fatty acids (SCFA; linear, P = 0.007) concentrations were elevated as the level of JA in the diet increased. Lactic acid content was increased (quadratic, P = 0.009) whereas pH (linear, P = 0.024 and quadratic, P = 0.047) and ammonia concentration (linear, P < 0.001) were reduced in faeces due to JA supplementation. In conclusion, feeding diets supplemented with JA tuber powder beneficially augments fibre utilization along with better apparent absorption of calcium and phosphorus and positive shift in large intestinal microbiota populations and SCFA concentrations.