Rumen ciliates are a specialized group of ciliates exclusively found in the anaerobic, carbohydrate-rich rumen microenvironment. However, the molecular and mechanistic basis of the physiological and behavioral adaptation of ciliates to the rumen microenvironment is undefined. We used single-cell transcriptome sequencing to explore the adaptive evolution of three rumen ciliates: two entodiniomorphids, Entodinium furca and Diplodinium dentatum; and one vestibuliferid, Isotricha intestinalis. We found that all three species are members of monophyletic orders within the class Litostomatea, with E. furca and D. dentatum in Entodiniomorphida and I. intestinalis in Vestibuliferida. The two entodiniomorphids might use H-2-producing mitochondria and the vestibuliferid might use anaerobic mitochondria to survive under strictly anaerobic conditions. Moreover, carbohydrate-active enzyme (CAZyme) genes were identified in all three species, including cellulases, hemicellulases, and pectinases. The evidence that all three species have acquired prokaryote-derived genes by horizontal gene transfer (HGT) to digest plant biomass includes a significant enrichment of gene ontology categories such as cell wall macromolecule catabolic process and carbohydrate catabolic process and the identification of genes in common between CAZyme and HGT groups. These findings suggest that HGT might be an important mechanism in the adaptive evolution of ciliates to the rumen microenvironment.