(−)‐Epigallocatechin gallate (EGCg), which has an antitumor activity, is a major component of polyphenol in green tea. Some mechanisms on the anticancer effect of EGCg have been proposed, and EGCg‐protein interaction is thought to be a significant step. However, the mechanisms on the regulation of protein function through binding are still unclear. To obtain the proof of functional modulation by EGCg, we used thiol enzymes, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and papain, as model proteins. Incubation of GAPDH with EGCg resulted in a dose‐dependent loss of the enzyme activity, which was accompanied by the loss of free thiol groups. Furthermore, by mass spectrometry (MS) and Western blotting with redox cycling staining, it is clarified that EGCg covalently binds to the enzyme. In addition, the results on the modification of papain were similar to that of GAPDH. To identify the binding site of GAPDH by EGCg, both the native and EGCg‐treated GAPDH were purified with catechol affinity resin and then digested with trypsin, and the resulting peptides were subjected to MS. We identified the EGCg‐binding peptides containing cysteine residues. These results suggest that modulation of GAPDH activity by EGCg is due to the selective modification of the cysteine residues in the GAPDH molecule. These findings may contribute to the understanding of anticancer mechanisms based on binding of tea catechins to proteins.