Protein as well as starch is fermented in the colon, but the interaction between protein and starch fermentation and the impact on colonic oncogenesis is unknown. High-protein diets increase delivery of protein to the colon and might promote oncogenesis through generation of toxic products. We investigated the interaction of resistant starch (RS) with digestion-resistant potato protein (PP) on colonic fermentation events and their relationship to intestinal tumourigenesis. Male Sprague-Dawley rats were fed an AIN-76A-based diet for 4 weeks and intestinal neoplasms were induced by azoxymethane. Experimental diets included the following: no added RS or PP, 10% high amylose maize starch (source of RS) replacing digestible starch, 15% PP replacing casein and 10% high amylose maize starch+15% PP. Rats were maintained on diets until killed at 30 weeks. Feeding RS significantly increased short-chain fatty acid (SCFA) levels (P<0.001) in the caecum and colon. Importantly, butyrate concentration was significantly increased in the distal colon with RS (P<0.001). Feeding PP increased protein fermentation products, but this effect was reduced by adding RS to the diet. Intestinal neoplasms and colorectal adenocarcinomas were reduced by feeding RS (P<0.01) regardless of whether PP was fed, whereas PP alone increased the incidence and number of small intestinal neoplasms including the adenocarcinomas (P<0.01). In conclusion, RS altered the colonic luminal environment by increasing the concentration of SCFAs including butyrate and lowering production of potentially toxic protein fermentation products. These effects of RS not only protected against intestinal tumourigenesis but also ameliorated the tumour-enhancing effects of feeding indigestible protein.