CONSORT Checklist.

<div> Metformin is a commonly used antihyperglycaemic agent for the treatment of type 2 diabetes mellitus. Nevertheless, the exact mechanisms of action, underlying the various therapeutic effects of metformin, remain elusive. The goal of this study was to evaluate the alterations in longitudinal whole-blood transcriptome profiles of healthy individuals after a one-week metformin intervention in order to identify the novel molecular targets and further prompt the discovery of predictive biomarkers of metformin response. Next generation sequencing-based transcriptome analysis revealed metformin-induced differential expression of genes involved in intestinal immune network for IgA production and cytokine-cytokine receptor interaction pathways. Significantly elevated faecal sIgA levels during administration of metformin, and its correlation with the expression of genes associated with immune response (<i>CXCR4</i>, <i>HLA-DQA1</i>, <i>MAP3K14</i>, <i>TNFRSF21</i>, <i>CCL4</i>, <i>ACVR1B</i>, <i>PF4</i>, <i>EPOR</i>, <i>CXCL8</i>) supports a novel hypothesis of strong association between metformin and intestinal immune system, and for the first time provide evidence for altered RNA expression as a contributing mechanism of metformin’s action. In addition to universal effects, 4 clusters of functionally related genes with a subject-specific differential expression were distinguished, including genes relevant to insulin production (<i>HNF1B</i>, <i>HNF1A</i>, <i>HNF4A</i>, <i>GCK</i>, <i>INS</i>, <i>NEUROD1</i>, <i>PAX4</i>, <i>PDX1</i>, <i>ABCC8</i>, <i>KCNJ11</i>) and cholesterol homeostasis (<i>APOB</i>, <i>LDLR</i>, <i>PCSK9</i>). This inter-individual variation of the metformin effect on the transcriptional regulation goes in line with well-known variability of the therapeutic response to the drug. </div>