Balantioides coli plays an important role in the diarrhea of weaned piglets, but its pathogenic potential and interaction with gut microbes remain unclear. To investigate the impact of B. coli colonization on the gut bacterial structure and function of weaned piglets, a metagenomic analysis based on shotgun sequencing was performed on fresh fecal samples collected from ten B. coli-colonized piglets and eight B. coli-free ones in this study. The results showed that decreasing diversity and shifted composition and function of the bacterial community were detected in the weaned piglets infected by B. coli. In contrast to the B. coli-negative group, the relative abundances of some members of the Firmicutes phylum including Clostridium, Ruminococcus species, and Intestinimonas butyriciproducens, which produce short-chain fatty acids, were significantly reduced in the B. coli-positive group. Notably, some species of the Prevotella genus (such as Prevotella sp. CAG:604 and Prevotella stercorea) were significantly increased in abundance in the B. coli-positive piglets. A functional analysis of the gut microbiota demonstrated that the differential gene sets for the metabolism of carbohydrates and amino acids were abundant in both groups, and the more enriched pathways in B. coli-infected piglets were associated with the sugar-specific phosphotransferase system (PTS) and the two-component regulatory system, as well as lipopolysaccharide (LPS) biosynthesis. Furthermore, several species of Prevotella were significantly positively correlated to the synthesis of lipid A, leading to the exporting of endotoxins and, thereby, inducing inflammation in the intestines of weaned piglets. Taken together, these findings revealed that colonization by B. coli was distinctly associated with the dysbiosis of gut bacterial structure and function in weaned piglets. Lower relative abundances of Clostridiaceae and Ruminococcaceae and higher abundances of Prevotella species were biomarkers of B. coli infection in weaned piglets.