Aims To evaluate efficacy of Saccharomyces cerevisiae fermentate prebiotic (EH) in protection of intestinal barrier integrity in rats during heat stress, to analyze the impact of heat stress and preventive treatment with EH on the structure of the gut microbiota. Methods and Results Two groups of rats were treated orally with EH or phosphate-buffered saline for 14 days. On day 15, half of the rats in each group were exposed to heat stress conditions, while control animals were kept at room temperature. Histological and Western blot analyses of the intestine, culture-based microbiological analysis and high-throughput 16S rRNA sequencing for the gut microbiota were performed for each rat. Exposure of animals to heat stress conditions resulted in inhibition of tight junction (TJ) proteins expression, decrease of Paneth and goblet cells, decrease of beneficial and increase of pathogenic bacteria. Oral treatment of rats with EH before stress significantly prevents these adverse effects by elevation of the gut beneficial bacteria, particularly butyrate-producing bacteria. Conclusions Essential effect of EH in protection of intestinal barrier integrity during heat stress is connected with beneficial modulation of the gut microbiota. Significance and Impact of the Study Our results will contribute to the development of new approaches to prevention of heat stress-related complications.