The considerable animal-to-animal variation in microbial profiles is a challenge in elucidating the role of gut microbiota in host metabolism. The main purpose of this study was, therefore, to develop a pig model with reduced animal-to-animal variation in gut microbial profile. Twelve piglets from four sows were reared conventionally and 12 piglets from four sows were reared artificially in high efficiency particulate air (HEPA) filtered isolators. All isolator-reared piglets were given an artificial colostrum formula containing the combined fecal material from all eight sows. All piglets were killed at 21 d of age and intestinal contents subjected to 16s rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) profiling. Resulting T-RFLP profiles clustered into two distinct groups representing the two treatment groups. Furthermore, Bray–Curtis dissimilarity distance values and Dice similarity indices showed reduced beta diversity in isolator-reared pigs indicating animal-to-animal variation was reduced in isolator-reared compared to conventional piglets. However, surprisingly, increased alpha diversity was observed in isolator-reared piglets compared with conventional piglets. In conclusion, the study demonstrated that rearing of piglets under conditions of controlled environment reduced animal-to-animal variation in the hindgut microbiota while paradoxically increasing within animal microbial diversity. Isolator rearing may be useful as a model to improve detection of treatment effects on gut microbiota.