Microsporidia are obligate intracellular eukaryotic parasites frequently associated with gastrointestinal disorders. As emerging eukaryotic microbes of clinical significance, understanding their interactions with host epithelial barriers is crucial for health maintenance. Short-chain fatty acids (SCFAs), key microbial metabolites produced by beneficial gut microbiota, play essential roles in maintaining intestinal epithelial integrity and modulating immune responses. This study investigates the potential impact of SCFAs on Microsporidia-induced epithelial barrier dysfunction in differentiated CACO-2. CACO-2 cells were cultured on transwell inserts to establish polarized intestinal epithelial monolayers. Infection severity was quantified by spore quantification from apical and basolateral compartments. Transepithelial electrical resistance (TEER) measurements assessed barrier integrity, while paracellular permeability was evaluated using fluorescent beads molecules. Cell viability assays determined cytotoxic effects of infection and impact of SCFA treatment. Additionally, Western blot analysis examined tight junction protein expression, immunofluorescence assays (IFA) visualized barrier architecture, and scratch assays assessed epithelial wound healing capacity. Results demonstrate that SCFA treatment significantly ameliorates Microsporidia-induced epithelial damage. SCFA-treated infected cells exhibited enhanced TEER values, reduced paracellular permeability, and improved cell viability. Western blot analysis revealed preserved tight junction protein levels, while IFA confirmed maintained physical barrier functionality. Scratch assays also demonstrated accelerated epithelial repair in SCFA-treated cells. Importantly, SCFA treatment reduced spore burden, suggesting direct anti-parasitic effects. These findings highlight the protective role of SCFAs in modulating eukaryotic microbe-host interactions, specifically in mitigating Microsporidia-induced epithelial dysfunction. Understanding these mechanisms provides insights into how beneficial microbial metabolites can counteract pathogenic eukaryotic microbes, with potential therapeutic implications for gut-related diseases.