Microorganisms rely on diverse ion transport and trace elements to sustain growth, development, and secondary metabolism. Manganese (Mn 2+ ) is essential for various biological processes and plays a crucial role in the metabolism of human cells, plants, and yeast. In Aspergillus flavus, we confirmed that Pmr1 localized in cis-and medial-Golgi compartments was critical in facilitating Mn 2+ transport, fungal growth, development, secondary metabolism, and glycosylation. In comparison to the wild type, the Δpmr1 mutant displayed heightened sensitivity to environmental stress, accompanied by inhibited synthesis of aflatoxin B1, kojic acid, and a substantial reduction in pathogenicity toward peanuts and maize. Interestingly, the addition of exogenous Mn 2+ effectively rectified the developmental and secondary metabolic defects in the Δpmr1 mutant. However, Mn 2+ supplement failed to restore the growth and development of the Δpmr1Δgdt1 double mutant, which indicated that the Gdt1 compensated for the functional deficiency of pmr1. In addition, our results showed that pmr1 knockout leads to an upregulation of O-glycosyl-Nacetylglucose (O-GlcNAc) and O-GlcNAc transferase (OGT), while Mn 2+ supplementation can restore the glycosylation in A. flavus. Collectively, this study indicates that the pmr1 regulates Mn 2+ via Golgi and maintains growth and metabolism functions of A. flavus through regulation of the glycosylation.