Oxygen limitation is regarded as a useful strategy to improve enzyme production by mycelial fungus like Aspergillus niger. However, the intracellular metabolic response of A. niger to oxygen limitation is still obscure. To address this, the metabolism of A. niger was studied using multi-omics integrated analysis based on the latest GEMs (genome-scale metabolic model), including metabolomics, fluxomics and transcriptomics. Upon sharp reduction of the oxygen supply, A. niger metabolism shifted to higher redox level status, as well as lower energy supply, down-regulation of genes for fatty acid synthesis and a rapid decrease of the specific growth rate. The gene expression of the glyoxylate bypass was activated, which was consistent with flux analysis using the A. niger GEMs iHL1210. The increasing flux of the glyoxylate bypass was assumed to reduce the NADH formation from TCA cycle and benefit maintenance of the cellular redox balance under hypoxic conditions. In addition, the relative fluxes of the EMP pathway were increased, which possibly relieved the energy demand for cell metabolism. The above multi-omics integrative analysis provided new insights on metabolic regulatory mechanisms of A. niger associated with enzyme production under oxygen-limited condition, which will benefit systematic design and optimization of the A. niger microbial cell factory.