To eliminate the feedback inhibition of acetohydroxyacid synthase (AHAS) by L-isoleucine and thereby improve L-isoleucine production, the mechanism by which feedback inhibition in AHAS is relieved was investigated. By adopting bioinformatic approaches, including structure prediction, molecular docking, and conservation analysis, key residue sites potentially involved in relieving product inhibition were identified. Virtual saturation mutagenesis and enzymological characterization were further used to select an optimal mutant, AHASR175L, which exhibited a 40% increase in specific activity compared to the wild-type enzyme. Subsequently, the engineered strain Cg.ILE-5-1 was constructed, which overexpressed the AHASR175L coding gene ilvBNR175L and the key enzyme gene ilvA. In a 5 L fermenter, this strain accumulated 27.84 g•L-1 of L-isoleucine, representing a 300% increase over shake-flask cultivation. The results demonstrate that the semi-rational engineering of AHASR175L effectively alleviated product inhibition and significantly enhanced L-isoleucine biosynthesis. This study provides a theoretical foundation for the biosynthesis of branched-chain amino acids.