Cronobacter sakazakii is an opportunistic foodborne pathogen causing intestinal and extra-intestinal diseases in humans, especially young children, and is regarded as one of the main concerns in public health. Saccharomyces boulardii is a well-known probiotic yeast widely used to treat and prevent antibiotic-associated diarrheal infections in infants and neonates. This study evaluated the preventive effects and potential of probiotic S. boulardii against C. sakazakii intestinal infections in humans. Viability, bacterial virulence factor, cellular pro-inflammatory gene expression, and nanomechanical properties of the cytoplasmic membrane of caco-2 cells were evaluated using MTT, real-time PCR, and AFM methods, respectively. Using histopathological analysis, S. boulardii treatment was evaluated on infected newborn C57 BL/6 mice. We found that S. boulardii inoculation significantly (P <
0.05) increased the viability and downregulated the cellular pro-inflammatory genes (IL-8 and NFkB) and bacterial virulence factor genes (ompA and hfq) in infected intestinal cells while also decreasing the morphological alterations. We also observed that S. boulardii treatment reduced the intestinal damage induced by C. sakazakii infection. In conclusion, our findings demonstrate that S. boulardii effectively protects against C. sakazakii infections. This probiotic yeast holds promise as a potential preventive and therapeutic agent for intestinal diseases associated with C. sakazakii.