Botryosphaeria dothidea is a pathogenic fungus that causes the brown spot disease in Gardenia jasminoides Ellis (GE). We carried out comprehensive research to reveal the mechanisms underlying leaf-mediated resistance to B. dothidea. First, we observed the ultrastructure of normal and B. dothidea-infested leaves through scanning and transmission electron microscopy. B. dothidea invaded the leaves mainly by lysing the epidermis, and the physiological structure of GE leaves are severely affected by B. dothidea infestation. Subsequently, we used RNA sequencing to elucidate the dynamic transcriptome of GE leaves 0 h, 12 h, 1 days, 2 days, and 3 days after infection with B. dothidea. The KEGG enrichment analysis based on differentially expressed genes revealed the most relevant pathways involved in defense, including phenylpropanoid biosynthesis, plant hormone signal transduction, and starch and sucrose metabolism pathways. Finally, the roles of key genes, such as COMT1, AUX1, and SUS3, which are involved in lignin synthesis, growth hormone transport, and cell wall formation, respectively, were identified through STRING database analysis and visualization with Cytoscape software. We used transcriptomics to analyze the mechanism of infestation of GE in response to a causal agent of brown spot disease (B. dothidea). These results provide novel insights into leaf-mediated stress and resistance of GE to diseases.