Cysteine, a common amino acid used in food, cosmetic, and pharmaceutical industries, has a growth inhibitory effect. This growth inhibition by cysteine poses a problem, as the production of cysteine using microbial cells results in decreased cell growth and cysteine productivity. The underlying mechanism of growth inhibition by cysteine is unclear. This study aims to understand the mechanism of growth inhibition by cysteine in Corynebacterium glutamicum. To do this, cysteine-resistant mutants of C. glutamicum were isolated based on adaptive laboratory evolution (ALE) and their characteristics were analyzed. Genome resequencing revealed that mutations in the open reading frame of the ilvN gene encoding the regulatory small subunit of acetohydroxyacid synthase (AHAS), which is involved in branched-chain amino acid biosynthesis, were found in ALE cell populations and the isolated cysteine-resistant mutants. The ilvN mutations which are responsible for increased valine production resulted in improved cell growth in the presence of cysteine. Moreover, the addition of valine to the culture medium mitigated growth inhibition by cysteine, whereas the addition of leucine and isoleucine showed a slight mitigation. Additionally, the activity of AHAS from C. glutamicum was inhibited by cysteine, whereas AHAS from the strains carrying ilvN mutations exhibited resistance to cysteine. These results indicate that growth inhibition by cysteine is caused by perturbations in the biosynthesis of branched-chain amino acids, particularly valine in C. glutamicum. Furthermore, the cysteine-resistant mutants obtained by ALE demonstrated enhanced cysteine production as production hosts, suggesting that cysteine resistance is a useful phenotype for cysteine production in C. glutamicum. KEY POINTS: • Cysteine-resistant mutants of C. glutamicum obtained by ALE were analyzed. • Perturbation of valine biosynthesis by cysteine results in growth inhibition in C. glutamicum. • Cysteine resistance is a useful phenotype for cysteine production by C. glutamicum.