Botryosphaeria dieback on the grapevine is caused by Botryosphaeriaceae fungi, which threatens the yield and quality of grapes. At present, chemical control strategies are often observed to be ineffective in controlling the disease worldwide. Improving our understanding of the molecular mechanisms that confer resistance to pathogens would facilitate the development of more pathogen-tolerant grape varieties. Here, we used RNA sequencing analysis to profile the transcriptome of grapevine green shoots infected with Lasiodiplodia theobromae over a time course of 4, 8 and 12 hours post inoculation. A total of 5181 genes were identified as differentially expressed genes (DEGs), and DEGs were more abundant over time. Further analysis revealed that many of these DEGs are involved in plant-pathogen interactions, hormone signal transduction and phenylpropanoid biosynthesis pathways, suggesting that innate immunity, phytohormone signaling and many phenylpropanoid compounds, which constitute a complex defense network in plants, are involved in the response of grapevine against to L. theobromae infection. This study provides novel insights into the molecular mechanisms of plant-pathogen interactions that will be valuable for the genetic improvement of grapevines.