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Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism on a molecular level could be an influence on miRNAs and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissue from Enterococcus faecium NCIMB 10415-fed piglets versus untreated controls using Next Generation Sequencing. We identified the miR-423-5p as highly affected by the treatment group (2.32 fold; P = 0.014). Validation by RT-qPCR confirmed a significant up-regulation of miR-423-5p (2.11 fold; P = 0.03) and, additionally a down-regulation of important immune-relevant target gene IGLC (0.61 fold; P = 0.03) and IGKC (0.69 fold; P = 0.04). Expression analysis of miR-423-5p and IGLC at different age points, show a clear anti-correlated relation. Luciferase reporter assays in a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding E. faecium on the expression of miR-423-5p and on the regulation of the immune globulin gene IGLC through miR-423-5p. This might be a possible mode of action of E. faecium on immune cell regulation in the small intestine.