Omics data was acquired, and the development and research of metabolic simulation and analysis methods using them were also actively carried out. However, it was a laborious task to acquire such data each time the medium composition, culture conditions, and target organism changed. Therefore, in this study, we aimed to extract and estimate important variables and necessary numbers for predicting metabolic flux distribution as the state of cell metabolism by flux sampling using a genome-scale metabolic model (GSM) and its analysis. Acetic acid production from glucose inEscherichia coliwith GSM iJO1366 was used as a case study. Flux sampling obtained by OptGP using 1000 pattern constraints on substrate, product, and growth fluxes produced a wider sample than the default case. The analysis also suggested that the fluxes of iron ions, O2, CO2, and NH4+, were important for predicting the metabolic flux distribution. Additionally, the comparison with the literature value of13C-MFA using CO2emission flux as an example of an important flux suggested that the important flux obtained by this method was valid for the prediction of flux distribution. In this way, the method of this research was useful for extracting variables that were important for predicting flux distribution, and as a result, the possibility of contributing to the reduction of measurement variables in experiments was suggested.