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 Δ pmr1 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 Δ pmr1 mutant. However, Mn 2+ supplement failed to restore the growth and development of the Δ pmr1 Δ gdt1 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-N-acetylglucose (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.