Adventitious root (AR) formation is important for the vegetative propagation. The effects of strigolactones (SLs) on AR formation have been rarely reported, especially in woody plants. In this study, we first verified the inhibitory effects of SLs on AR formation in apple materials. Transcriptome analysis identified 12,051 differentially expressed genes over the course of AR formation, with functions related to organogenesis, cell wall biogenesis or plant senescence. WGCNA suggests SLs might inhibit AR formation through repressing the expression of two core hub genes, MdLAC3 and MdORE1. We further verified that enhanced cell wall formation and accelerated senescence were involved in the AR inhibition caused by SLs. Combining small RNA and degradome sequencing, as well as a dual-luciferase sensor system, we identified and validated three negatively correlated miRNA–mRNA pairs, including mdm-miR397–MdLAC3 involved in secondary cell wall formation, and mdm-miR164a/b–MdORE1 involved in senescence. Finally, we have experimentally demonstrated the role of mdm-miR164b–MdORE1 in SLs-mediated inhibition of AR formation. Overall, our findings not only propose a comprehensive regulatory network for the function of SLs on AR formation, but also provide novel candidate genes for the potential genetic improvement of AR formation in woody plants using transgenic or CRISPR technology.