The aim of this study was to investigate the effects of a low dietary protein and high carbohydrate infant formula on the large intestine of neonatal rats. A total of 24 neonatal Sprague-Dawley rats (14-days-old) were randomly assigned to the low protein, high carbohydrate infant formula-fed group (I group) and a human breast milk-fed group (H group). After 7 days, we selected 6 rats at random from each group to study. No significantly different microbial colonization patterns were observed in the 2 groups at the phylum level. At the family level, Enterobacteriaceae and Bacteroidaceae were the dominant bacteria in I and H rats. While Bacteroides was the most abundant bacteria at the genus level, no significant difference was observed between the 2 groups. Methanoic acid, acetate, and butyrate increased in concentration in the I group compared with the H group. Protease activities, ammonia, and indole in the large intestine were lower in I rats than H rats. A significant increase in the expression of GADPH and decrease in the expression of aquaporin 8, aminopeptidase A, cathepsin F precursor, and ubiquitin carboxyl-terminal hydrolase FAF-Y were observed in I rats compared with H rats. These results suggest that a low protein diet could modulate the microbial ecology in the large intestine of neonatal rats.