L-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of L-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in L-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in L-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to L-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for L-cysteine tolerance in E. coli cells. However, L-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent L-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of L-cysteine, the transformants exhibited more L-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in L-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for L-cysteine production in E. coli.