The phyllosphere is one of the largest habitats for microorganisms, and host genetic factors play an important role during the interaction between microorganisms and the phyllosphere. Therefore, the transgene may also lead to changes in the maize phyllosphere. ZmMYB3R was identified as a drought-tolerant gene in Arabisopsis. Here, we employed metagenomic sequencing to analyze the microbiome of the adaxial and abaxial leaf surfaces on ZmMYB3R-overexpressing (OE) and wild-type (WT)·maize, aiming to dissect the possible associations between ZmMYB3R and changes in phyllosphere microbiome functioning. Our results revealed that overexpressing ZmMYB3R altered the alpha and beta diversity of the phyllosphere microbiome. In OE plants, more beneficial microbes accumulated on the phyllosphere, while pathogenic ones diminished, especially on the abaxial surface of ZmMYB3R leaves. Further analysis of disease resistance-related metabolic pathways and abundances of disease resistance genes revealed significant differences between OE and WT. The inoculation experiment between OE and WT proved that ZmMYB3R increased the disease resistance of maize. In conclusion, the results reveal that transgenes affect the phyllosphere microbiome, and ZmMYB3R might alter leaf disease resistance by reshaping the phyllosphere microbiome structure. These findings help us understand how ZmMYB3R regulates leaf disease resistance and may facilitate the development of disease control by harnessing beneficial microbial communities.