Considering of the possible application of fatty acids secretion in industrial production process for reducing extraction costs, the effects of enhanced fatty acids secretion by knocking out acyl-CoA synthetases on the global regulation, cellular metabolism were investigated through integrated omics analysis. Firstly, functional category analysis of differential expression genes showed that most of the genes were associated with the cell cycle and biology process such as cellular component biogenesis, cell wall organization and so on. Secondly, the decreased mRNA levels of Tdh, Eno and Pgk1 in glycolysis in faa mutant YB526 were confirmed by the differential protein analysis. As described in previous reports, the decreased protein levels of Tdh, Eno and Pgk1 were resulted from the increase of 14-3-3 protein activity. However, the increased level of 14-3-3 proteins could not improve the biosynthesis of ergosterol in the acyl-CoA deficiency strain YB526 due to the toxic effects of fatty acids. Thirdly, the decrease of ergosterol and ceramide contents in cell envelopes can improve the permeation of membrane and fatty acids secretion in faa mutant YB526. This comprehensive omics analysis could be helpful in the rational design of robust yeasts for the production of free fatty acids or other biobased chemicals.