Grapevine Trunk Diseases (GTDs) are among the most destructive diseases in viticulture due to global climate change. Some causal agents like Neofusicoccum parvum can be latent endophytic and become pathogenic under abiotic stress. Microtubules (MTs) have been found to play a role in mediating the pathogen-related signaling in grapevine. In this study, a novel transcription factor VrWRKY22 was identified and cloned from the native American grapevine Vitis rupestris. Leaves of the table grape variety ‘Kyoho’ ( V. vinifera × V. labrusca L.) overexpressing VrWRKY22 showed less necroses after N. parvum Bt‐67 inoculation and activated signaling pathways. VrWRKY22 was phosphorylated by VrMPK3 and then bound to the TTGACC motif in the promoter of VrKIN10C, which was confirmed by Y2H and Y1H assays. Since VrKIN10C is one of the important kinesin-like proteins associated with microtubules, a grapevine MT marker line overexpressing VrWRKY22 was generated to test the responses of grapevine cells to N. parvum Bt‐67. An increased number of prompt movement proteins can be traced within the peri-nuclear MTs and along the cortical MTs. The skewness and thickness of both central and cortical MTs were significantly increased. Moreover, a prominent (resulting from both the number and the rate) accumulation of speckles appeared in the nucleus and cortical MTs. A significant reduction in cell mortality and a stronger antioxidant capacity were detected. This study demonstrates that VrWRKY22 plays positive roles during N. parvum Bt‐67 invasion by rapidly increasing the concentration and dynamics of MTs in the peri-nuclear and cortical regions via VrKIN10, and will facilitate the interpretation of the results of further GTD mitigation studies.