Ergosterol, a major lipid present in the fungal cell membrane, is considered as an effective antifungal drug target. A rational strategy for increasing drug reservoir relies on functionally validation of essential enzymes involved in fungal key biological pathway. Current knowledge regarding the essential genes in the ergosterol biosynthesis pathway is still limited in the opportunistic human pathogen Aspergillus fumigatus. In this study, we characterized two endoplasmic reticulum-localized sterol C-14 reductases encoded by both erg24A and erg24B homologs that are essential for the viability of A. fumigatus despite the fact that neither paralog is essential individually. Loss of one homolog of Erg24 impairs hyphal growth, conidiation, and virulence but has no effect on ergosterol biosynthesis. To investigate the functional significance of erg24, a conditional double mutant (Delta erg24B niiA::erg24A) was constructed in the Delta erg24B background. Strikingly, the conditional erg24 double mutant exhibited severe growth defects and accumulation of sterol intermediate. Moreover, the addition of metal ions and the overexpression of the corresponding ion transporters could rescue the growth defects of the erg24 double mutant in A. fumigatus, implying that the defective phenotype of the erg24 double mutant is tightly associated with dysregulation of ion homeostasis. Taken together, our results demonstrate the critical role of Erg24 in ergosterol biosynthesis and ion homeostasis in A. fumigatus, which may have important implications for antifungal discovery.