ABSTRACT We recently demonstrated that cow's milk fermented with the probiotic Lactobacillus paracasei CBA L74 (FM-CBAL74) reduces the incidence of respiratory and gastrointestinal tract infections in young children attending school. This effect apparently derives from a complex regulation of non-immune and immune protective mechanisms. We investigated whether FM-CBAL74 could regulate gut microbiota composition and butyrate production. We randomly selected 20 healthy children (12 to 48 months) from the previous randomized controlled trial, before (t0) and after 3 months (t3) of dietary treatment with FM-CBAL74 (FM) or placebo (PL). Fecal microbiota was profiled using 16S rRNA gene amplicon sequencing, and the fecal butyrate concentration was also measured. Microbial alpha and beta diversities were not significantly different between groups prior to treatment. FM-CBAL74 but not PL treatment increased the relative abundance of Lactobacillus . Individual Blautia , Roseburia , and Faecalibacterium oligotypes were associated with FM-CBAL74 treatment and demonstrated correlative associations with immune biomarkers. Accordingly, PICRUSt analysis predicted an increase in the proportion of genes involved in butyrate production pathways, consistent with an increase in fecal butyrate observed only in the FM group. Dietary supplementation with FM-CBAL74 induces specific signatures in gut microbiota composition and stimulates butyrate production. These effects are associated with changes in innate and acquired immunity. IMPORTANCE The use of a fermented milk product containing the heat-killed probiotic strain Lactobacillus paracasei CBAL74 induces changes in the gut microbiota, promoting the development of butyrate producers. These changes in the gut microbiota composition correlate with increased levels of innate and acquired immunity biomarkers.