To analyse the molecular mechanisms of phytoplasma pathogenicity, the comprehensive metabolomic changes of mulberry leaf and phloem sap in response to phytoplasma infection were examined using gas chromatography-mass spectrometry. The metabolic profiles obtained revealed that the metabolite compositions of leaf and phloem sap were different, and phytoplasma infection has a greater impact on the metabolome of phloem sap than of leaf. Phytoplasma infection brought about the content changes in various metabolites, such as carbohydrates, amino acids, organic acids, etc. Meanwhile, the results of biochemical analysis showed that the degradation of starch was repressed, and the starch content was increased in the infected leaves. In addition, we found that phytoplasma infection changed the levels of abscisic acid and cytokinin and break phytohormone balance. Interestingly, our data showed that the contents of H2O2 and superoxide were increased in the infected leaves, but not in the phloem saps. Based on the results, the expression levels of the genes involved in the metabolism of some changed metabolites were examined, and the potential molecular mechanisms of these changes were discussed. It can be concluded that both the leaf and phloem saps have a complicated metabolic response to phytoplasma infection, but their response mechanisms were different. A multi target metabolite profiling was characterized by GC/MS to provide for the first time a global analysis of the changes that occur in mulberry following infection by yellows dwarf phytoplasma. Together with global metabolite analysis, some gene expressions and biochemical changes were analyzed, and the potential molecular mechanisms of these changes were discussed. It was pointed out that both the leaf and phloem sap have a complicated metabolic response to phytoplasma infection, but their metabolic changes and response mechanisms were different.