The storage of spent maitake culture medium (SMCM) under various conditions was investigated as a potential pretreatment of SMCM for increased ethanol conversion. When SMCM was stored at 25°C for 12 weeks, the glucose yield by enzymatic saccharification increased from 22 to 52%. Selective degradation of lignin and hemicellulose occurred in SMCM during storage. The optimal storage temperature of SMCM was at the active growing temperature of maitake mycelium, which is between 25 and 30°C. Storage of SMCM under anaerobic conditions did not increase the glucose yield compared to non-stored conditions. The glucose yield from the SMCM stored for 4 weeks at 25°C was increased by about 30% with either NaOH or vibrating ball milling pretreatment. After 12 weeks of storage, the glucose yield from SMCM without any other pretreatment was higher than that of non-stored SMCM with additional pretreatments. An ethanol yield of 42.1% was obtained from SMCM stored for 12 weeks by simultaneous saccharification and fermentation, which was comparable to yields after NaOH (41.3%) or vibrating ball milling (44.1%) pretreatments. Therefore, storage of SMCM is a very useful pretreatment for bioethanol production.