Pigeons, as the only altricial birds in poultry, are the primary Trichomonas gallinae (T. gallinae) host. To study the effects of T. gallinae infection on gut microbiota, we compared the microbiota diversity and composition in gastrointestinal (GI) tracts of pigeons at the age of 14 and 21 day with different degrees of T. gallinae infection. Thirty-six nestling pigeons were divided into three groups: the healthy group, low-grade and high-grade trichomonosis group. Then, the crop, small intestine and rectum contents were obtained for sequencing of the 16S rRNA gene V3-V4 hypervariable region. The results showed that the microbiota diversity was higher in crop than in small intestine and rectum, and the abundance of Lactobacillus genus was dominant in small intestine and rectum of healthy pigeons at 21 days. T. gallinae infection decreased the microbiota richness in crop at 14 days. The abundance of the Firmicutes phylum and Lactobacillus genus in small intestine of birds at 21 days were decreased by infection, however the abundances of Proteobacteria phylum and Enterococcus, Atopobium, Roseburia, Aeriscardovia and Peptostreptococcus genus increased. The above results indicated that crop had the highest microbiota diversity among GI tract of pigeons, and the gut microbiota diversity and composition of pigeon squabs were altered by T. gallinae infection.