Zymoseptoria tritici is one of the most economically destructive wheat diseases all over the world and is a model fungal plant pathogen within the ascomycetes. In this study, the instrumental role of the ZtRlm1 gene encoding a MADS-box transcription factor (TF) in the infection process of Z. tritici was functionally characterized as these proteins play critical roles in the global gene regulation required for various developmental and physiological processes. Our infection assays showed that ZtRlm1 mutants were attenuated in disease development as a 30 and 90% reduction in chloro-necrotic lesions and pycnidia formation, respectively, were observed in plants inoculated with ZtRlm1 mutant strains demonstrating that ZtRlm1 is a crucial factor playing a significant role in the late stage of infection corresponding with pycnidial formation. Our expression analysis demonstrated that the transcript level of ZtRlm1 is induced at 2 and 20 days post-inoculation, coinciding with pycnidial sporulation. In addition, microscopic analyses showed that branch intensity and biomass production were significantly reduced, indicating that impaired pycnidia formation is a result of impaired differentiation and biomass production in the ZtRlm1 mutants. Furthermore, melanization, a phenomenon required for fruiting body formation, was significantly hampered in ZtRlm1 mutants as they were not melanized under all tested temperature and media conditions. Overall, our data showed that impaired disease development of the ZtRlm1 mutants is mainly due to the significant impact of ZtRlm1 in different cellular processes, including differentiation, branching, fungal biomass production, and melanization, in which identification of downstream genes are of interest to increase our understanding of this pathosystem.