The spent mushroom substrate (SMS) generated by mushroom production is applicable to the anaerobic digestion process. However, SMS-anaerobic digestion residue (ADR) can be produced. In this study, SMS-ADR was used as a medium base material for Enokitake (Flammulina velutipes) mushroom cultivation to examine the potential for food production. Two types of pretreated SMS-ADR prior to the anaerobic digestion process-KOH pretreated (KOH-treated SMS-ADR), and NaOH pretreated (NaOH-treated SMS-ADR) were carried out in this study. The KOH- and NaOH-treated SMS-ADR contained 1340.6 mg/100 g-Dry Matter (D.M.) of K, and 941.2 mg/100 g-D.M. of Na, respectively, indicating a large inorganic component derived from the chemicals used for pretreatment. The yields were 244.1 ± 3.7 g-Fresh Matter (F.M.)/bottle (10.5 ± 0.2 g-F.M./10 g medium-D.M.) for KOH-treated SMS-ADR with 75% and 255.5 ± 4.3 g-F.M./bottle (11.7 ± 0.5 g-F.M./10 g medium-D.M.) for NaOH-treated SMS-ADR with 50% as medium base material, which were closed to the standard medium. In KOH- and NaOH-treated SMS-ADR medium, the spread of the fungus on the 29th day of culture was more than 90%, suggesting sufficient mycelial growth. The amounts of general components, inorganic components and free amino acids in the fruiting bodies were the same as those obtained from the standard medium. In addition, we prepared the testing section, where SMS not applied to the anaerobic digestion process was used as the medium base material, and the inhibition of mycelial growth and fruiting bodies were observed.