Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation.