In this study, a genome-scale metabolic model (iCY1106) for Mortierella alpina ATCC 32222 was successfully reconstructed using knowledge from the scientific literature and publicly accessible databases. The model comprised 1106 genes, 1854 reactions and 1732 metabolites, and included 247 transport reactions and 216 exchange reactions. Following a series of simulations and verification by measurement of growth rates and substrate usage, the model was found to agree with published literature. The model was used to investigate the effects of the important precursors acetyl-CoA and NADPH on the biosynthesis of PUFAs such as ARA and EPA. FBA results showed that enhancing the pyruvate dehydrogenase complex increased acetyl-CoA availability for increasing ARA production. Limiting the nitrogen source was an effective method for reducing acetyl-CoA consumption, and malic enzyme was found to be a key node in the regulation of NADPH in ARA biosynthesis. Model iCY1106 could serve as a useful predictive tool for future systems biology studies to guide the genetic engineering of M. alpina to improve the production of industrially important metabolites.