► GGT and C-S lyase from Lentinula edodes were purified and characterised. ► Both GGT and C-S lyase caused the generation of endogenous formaldehyde. ► GGT was composed of subunits of 41 and 25kDa, C-S lyase was a homodimer of 46kDa. ► GGT was optimally active at pH 8.8/37°C, stable at pH 8.0–10.0/20–50°C. ► C-S lyase was optimally active at pH 8.5/40°C, stable at pH 8.0–9.0/20–60°C. In this study, γ-glutamyl transpeptidase (GGT) and l-cysteine sulphoxide lyase (C-S lyase) were purified from the fruiting body of Lentinula edodes in three steps and then characterised. We found that GGT together with C-S lyase caused the generation of endogenous formaldehyde in L. edodes. GGT was composed of a large subunit of 41kDa and a small subunit of 25kDa, and C-S lyase was composed of two identical subunits of 46kDa, as determined by SDS–PAGE. GGT was stable at pH 8.0–10.0 with an optimum pH of 8.8, and was stable at 20–50°C with an optimum activity at 37°C. C-S lyase was stable at pH 8.0–9.0 with an optimum pH of 8.5, and was stable at 20–60°C with an optimum activity at 40°C. The present work supports the study of the mechanism of endogenous formaldehyde in L. edodes.