Rooting and root development in Acer rubrum have important effects on overall growth. A. rubrum does not take root easily in natural conditions. In this study, the mechanisms of the miR396b- GRF1 module underlying rooting regulation in A. rubrum were studied. The subcellular localization and transcriptional activation of miR396b and its target gene growth regulating factor 1 ( GRF1 ) were investigated. These experiments showed that GRF1 was localized in the nucleus and had transcriptional activation activity. Functional validation experiments in transgenic plants demonstrated that overexpression of Ar- miR396b inhibited adventitious root growth, whereas overexpression of ArGRF1 increased adventitious root growth. These results help clarify the molecular regulatory mechanisms underlying adventitious root growth in A. rubrum and provide some new insights into the rooting rate in this species.