The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.