The effect of sucrose and resistant starch (‘CrystaLean’– a retrograded, amylose starch) on human gut microflora and associated parameters was studied in human flora‐associated (HFA) rats, colonized with microfloras from UK or Italian subjects, to determine whether such floras were affected differently by dietary carbohydrates. Consumption of the resistant starch diet resulted in significant changes in four of the seven main groups of bacteria enumerated. In both the UK and Italian flora‐associated rats, numbers of lactobacilli and bifidobacteria were increased 10–100‐fold, and there was a concomitant decrease in enterobacteria when compared with sucrose‐fed rats. The induced changes in caecal microflora of both HFA rat groups were reflected in changes in bacterial enzyme activities and caecal ammonia concentration. Although it had little effect on caecal short‐chain fatty acid concentration, CrystaLean markedly increased the proportion of n‐butyric acid in both rat groups and was associated with a significant increase in cell proliferation in the proximal colon of the Italian flora‐associated rats. CrystaLean appeared to play a protective role in the colon environment, lowering caecal ammonia concentration, caecal pH and β‐glucuronidase activity.