Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faecium NCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR- 423-5p as being greatly affected by the treatment group (2.32-fold; P=0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold; P=0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold; P=0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold; P=0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anticorrelated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding of E. faecium on the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action of E. faecium on immune cell regulation in the small intestine.