Mikania micrantha is one of the 100 worst invasive species globally and can cause significant negative impacts to agricultural and forestry economics, particularly in Asia and the Pacific region. The rust Puccinia spegazzinii has been used successfully as a biological control agent in several countries to help manage M. micrantha. However, the response mechanisms of M. micrantha to P. spegazzinii infection have never been studied. To investigate the response of M. micrantha to infection by P. spegazzinii, an integrated analysis of metabolomics and transcriptomics was performed. The levels of 101 metabolites, including organic acids, amino acids and secondary metabolites in M. micrantha infected with P. spegazzinii, were significantly different compared to those in plants that were not infected. The relative amounts of L-leucine, L-ioleucine, L-tryptophan, histidine, L-phenylalanine and L-citrulline were increased under P. spegazzinii infection. These may convert to intermediates within the TCA cycle, participating in energy biosynthesis. In addition, phytoalexins, such as maackiain, nobiletin, vasicin, arachidonic acid and JA-Ile, were synthesized and accumulated in M. micrantha in response to P. spegazzinii infection. A total of 4,978 differentially expressed genes were identified in M. micrantha infected by P. spegazzinii. Many key genes of the Mitogen-activated protein kinase (MAPK) signalling pathway, showed significantly higher expression under P. spegazzinii infection. These results not only help us to understand the response of metabolites and gene expression in M. micrantha following infection by P. spegazzinii, but also help us understand the potential for the continuous biological control of M. micrantha using P. spegazzinii.