Phoma macdonaldii causes black stem in sunflower, which severely affects both the yield and quality of sunflowers including weight and oil content of seeds. In this study, a global proteomic analysis of P. macdonaldii was performed in order to facilitate a better understand of the biological and pathogenic characteristics of this fungus. A total of 1,498 proteins were identified by LC-MS/MS in all biological replicates. The identified proteins were classified into functional categories, which included biological process, cellular components and molecular function (GO analysis), metabolism, genetic information progressing, environmental information progressing, cellular processes and organismal systems (KEGG). In addition, the expression of eight genes associated with fungal development and pathogenicity was further investigated. To the best of our knowledge, this is the first study to characterize the proteome of P. macdonaldii. Results from this study provide novel insights into fungal development and pathogenesis of P. macdonaldii and, perhaps even other Phoma species and may contribute to an enhanced understanding the sunflower and P. macdonaldii pathosystem