New evidence reveals that bol-miR159, an miRNA rich in fruits and vegetables, cross-kingdomly functions in mammalian bodies. However, whether the miRNA could regulate gut microbiota remains unclear. Here, the effect of miR159 on mouse intestinal microbes was comprehensively examined. The results showed that supplementation of miR159 to the chow diet significantly enhanced the diversity of mouse gut microbiota without causing pathological lesions or inflammatory responses on the intestines. At the phylum level, miR159 increased the abundance of Proteobacteria and decreased the Firmicute-to-Bacteroidetes (F/B) ratio. miR159 had prebiotic-like effects on mouse gut microbiota, as it promoted the growth of the bacteria that is beneficial for maintaining gut health. The miRNA can target bacteria genes and get into the bacteria cells. The data provide direct evidence on the crosstalk between plant miRNAs and intestinal microbes, highlighting the potential for miRNA-based strategies that modulate gut microbes to improve host health.