Annealing and heat-moisture treatment (HMT) are shown to be suitable methods to increase the yield of resistant starch type 3 (RS3) from Novelose 330 by up to 75%. Peak temperatures of approximately 121°C were used to produce to a sufficiently high thermal stability of the hydrothermal modified RS3 products for a wide range of applications. HMT significantly increased the crystallinity up to 40%. An in vivo feeding experiment with Wistar rats showed that fermentation of Novelose 330 dominated in the proximal colon, but degradation of HMT-Novelose was more dominant in the distal colon, leading to higher butyrate concentrations in this segment of the large bowel. Large-bowel surface and crypt length increased in the proximal colon in rats fed the Novelose 330-containing diet. In contrast, after the intake of HMT-Novelose, maximal values were found in the distal segment. The lower pH and higher butyrate concentration of the caecal and colonic contents significantlysuppressed the formation of secondary bile acids in RS3-fed rats. The formation of secondary bile acids was inhibited more strongly by HMT-Novelose than by Novelose 330. The Ki-67-immunopositive epithelial cells in the colon of RS3-fed rats indicated the establishment of an optimalbalance in the dynamic process of mucosal regeneration. HMT provides a method for the economical production of a high-quality RS3 with dominating prebiotic properties in the distal colon for health-promoting applications.