Simple SummaryBalantioides coliis a zoonotic parasite with a global distribution that inhabits the cecum and colon of humans and animals. This pathogen frequently causes diarrhea in weaned piglets undergoing the stress of transitioning from sow milk to pellet feed containing starch, leading to significant economic losses in the swine industry. Starch is necessary for the growth of the ciliated parasite. Here, our study revealed thatB. coliis able to effectively consume and digest starch into glucose moieties. The molecular mechanism of the effect of starch on the growth and proliferation ofB. coliby promoting the cell cycle and suppressing the autophagy of trophozoites has been elucidated. These findings will provide a novel insight to the pathogenesis of balantidial diarrhea in weaned piglets and an important reference for the control of balantiosis via adjusting the starch level in pellet feed.AbstractCarbohydrates are the main source of nutrition forB. coli, supplying energy for cell growth and development. The research aimed at investigating the mechanism of starch on the growth and replication ofB. coli. Single-cell separation was used to isolate single trophozoites ofB. coliunder a stereomicroscope, transcriptomic profiling was conducted based on the SMART-seq2 single-cell RNA-seq method. Comparative genomic analysis was performed onB. coliand eight other ciliates to obtain specific and expanded gene families ofB. coli. GO and KEGG enrichment analysis were used to analyze the key genes ofB. coliunder the action of starch in the present study. The results of single-cell RNA-seq depicts starch affected the growth and replication ofB. coliin two ways: (1) the cell cycle was positively promoted by the activation of the cAMP/PKA signaling pathway via glycolysis; (2) the cell autophagy was suppressed through the PI3K/AKT/mTOR pathway. Genes involved in endocytosis, carbohydrate utilization, and the cAMP/PKA signaling pathway were highly enriched in both specific and expanded gene families ofB. coli. Starch can be ingested and hydrolyzed into glucose, in turn affecting various biological processes ofB. coli. The molecular mechanism of the effect of starch on the growth and proliferation ofB. coliby promoting cell cycle and inhibiting the autophagy of trophozoites has been elucidated in our study.