In horses, the gut microbiota plays a critical role in maintaining intestinal homeostasis and overall health, but antimicrobial administration can disrupt its balance, resulting in dysbiosis, which can cause antibiotic-associated diarrhea. This study assessed the impact of intravenous cephalothin sodium administration on the equine gut microbiota by using microbial amplicon sequencing targeting the 16S-ITS-23S rRNA operon. Fecal samples from 39 clinically normal horses, 16 antibiotic-treated horses (cephalothin, n=5; minocycline, n=5; metronidazole, n=6, each administered for 5 days), and seven colitis-affected horses were collected. Cephalothin had minimal effects on microbial diversity, as α-diversity and β-diversity did not differ significantly from those in clinically normal horses (P>0.05). In contrast, minocycline and metronidazole significantly disrupted the microbial composition. Colitis-affected horses exhibited severe microbial alterations, including reduced species richness. Among the 52 microbial families significantly altered in colitis horses, none showed significant changes in response to cephalothin, suggesting its gut-microbiota-friendly characteristics. Notably, Verrucomicrobia subdivision 3, associated with mucosal integrity, remained stable in the cephalothin group, whereas its relative abundance significantly decreased in the minocycline, metronidazole, and colitis groups (P<0.05). These findings suggest that cephalothin poses a lower risk of equine microbiota disruption than other antimicrobials, making it a safer option in veterinary practice. Cephalothin could help to maintain gut microbial balance while ensuring effective infection control and reducing gastrointestinal complications. This study provides valuable evidence for optimizing antimicrobial selection, supporting equine gut microbiota health.