Purine biosynthesis serves vital roles in sustaining life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As this metabolic process is an integral determinant of fungal survival and virulence, enzymes within this pathway have been pursued as potential antifungal drug targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in several clinically prominent fungal pathogens, including Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. However, the lack of structural information for GMP synthase has hindered drug design efforts. In order to address this gap in knowledge, we have determined the crystal structure of A. fumigatus GMP synthase (2.3 Å), the first of fungal origin. Structural analyses of fungal GMP synthase reveal a distinct absence of the D1 dimerisation domain which is present in the human homologue. Interestingly, GMP synthase from A. fumigatus adopts a dimeric state, as determined by native mass spectrometry and gel filtration chromatography, which is contrary to the monomeric human homologue. Analysis of the substrate binding pockets of GMP synthase reveals key differences in the ATP and XMP binding site that can be exploited for species-specific inhibitor design. Furthermore, we have demonstrated the inhibitory activity of the glutamine analogues, acivicin (IC50 = 16.6 ± 2.4 µM) and 6-diazo-5-oxo-L-norleucine (IC50 = 29.6 ± 5.6 µM) against A. fumigatus GMP synthase. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug discovery endeavours.