The gut microbiota plays a crucial role in maintaining the host's health by enhancing immune function and resisting foreign pathogens. However, there remains a lack of comprehensive understanding regarding age-related alterations in the gut microbiota of equines. DNA was extracted from fecal samples collected from juvenile and middle-aged equines, followed by comprehensive data analysis, integration, and refinement. Subsequently, diversity analysis, taxonomic classification, and differential analysis were conducted to investigate the differences in gut microbiota between middle-aged and juvenile equines. The composition and diversity of the intestinal microbiome in middle-aged equines differs significantly from that of juvenile equines. There is an increase in species diversity within the Firmicutes, Verrucomicrobia, Anaerovorax, and Akkermansia phyla, while a decrease is observed in the Bacteroidota, Proteobacteria, Empedobacter, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium phyla. The population of Actinobacteria exhibits a significant increase in middle-aged equine compared to that observed in juvenile equines. Additionally, functional prediction analysis reveals enrichment of outer membrane receptor proteins (mostly Fe transport), AcrA subunit of multidrug resistance transporter (membrane fusion protein), and dipeptidyl aminopeptidase/carboxypeptidase in differential microbiomes. The aging process may induce modifications in the composition of intestinal microbiota, thereby influencing iron transporters, multidrug-resistant transporters, and transcriptional regulatory factors. The enhanced presence of Actinobacteria has the potential to modulate the susceptibility of middle-aged equines to diseases through regulation of iron transport proteins and multidrug resistance efflux pumps. These findings provide a theoretical foundation for further exploration into the impact of age on the compositional characteristics of the equine intestinal microbiome.