Mikania micrantha Kunth is a fast-growing global invasive weed species that causes severe damage to natural ecosystems and very large economic losses of forest and crop production. Although Puccinia spegazzinii can effectively inhibit the growth of M. micrantha and is used as a biological control strain in many countries, the mechanism of inhibiting the growth in Mikania micrantha is not clear. Here, we used a combination of phenotypic, enzyme activity, transcriptomic and metabolomic approaches to study the response of M. micrantha after infection by P. spegazzinii. In the early stages of rust infection, JA, JA-Ile and SA levels in infected leaves were significantly lower than that in uninfected leaves. In teliospore initial and developed stages of P. spegazzinii, JA and JA-Ile levels substantially increased by more than 6 times, which resulted in a significant decrease in accumulation of defense hormone SA in infected leaves of M. micrantha. The contents of plant growth-promoting hormones were significantly reduced in the infected plants as a result of substantial downregulation of expression of key genes related to hormone biosynthesis. Furthermore, rust infection led to high levels of ROS in chloroplasts and the destruction of chlorophyll structure, which also led to decreased photosynthetic gene expression, net photosynthetic rate, activity of Rubisco and levels of important organic acids in the Calvin cycle. We hypothesised that after P. spegazzinii infection, JA or JA-Ile accumulation not only inhibited SA levels to promote rust infection and development, but also impeded the rapid growth of M. micrantha by affecting plant growth hormones, carbon, and nitrogen metabolic pathways.