Over the past few decades, food allergy has continued to rise, significantly affecting our health, economy, and quality of life. However, current therapeutic strategies have limited efficacy and need to be improved. One alternative to prevent or reduce allergies is to modulate immunity and microbiota. Human milk (HM) could be considered a protective factor against food allergy, but how probiotics in human milk impact the susceptibility to food allergy remains unknown. Therefore, we studied the preventive impact of human milk Lactobacillus rhamnosus Probio-M9 on food allergy in ovalbumin (OVA)-sensitized mice. We studied the effects of oral administration of Probio-M9 on allergic signatures, immune response, gut microbiota, and metabolism. Oral therapeutic administration of live Probio-M9, but not heat-killed Probio-M9, significantly reduces OVA-specific IgE (OVA-sIgE), histamine, and mMCP-1 (mouse mast cell protease-1) levels in OVA-sensitized mice. Moreover, Probio-M9 supplementation reduced allergic inflammation and changes in the Th2/Th1 balance toward a dampened Th2 response. 16S rDNA sequencing analysis revealed an increased ratio of Firmicutes/Bacteroidota (F/B) and the relative abundance of short-chain fatty acid (SCFA)-producing Clostridia in the feces after Probio-M9 intake. Simultaneously, Probio-M9 significantly increased the levels of SCFAs and promoted the phosphorylation of signal transducer and activator of transcription 3 (STAT3), thereby inducing the expression of the antimicrobial peptides (AMPs) Reg3b and Reg3g. Our findings suggest that the use of Probio-M9 can be a potent strategy in food allergy prevention.