Abstract: Using the commonly applied protein evaluation methods, the nutritionalqualityof proteins in the fruiting bodies of Flammulina velutipes cultivated under simulated microgravity was compared with that of the mushrooms grown under regular condition (control). It was found that 14 amino acids (AA)in the mushrooms under treatment were higher than control. The contents of total AA and essential AA were 151.2 g·kg^-1 (i.e., 26.32% increase over control) and 81.7 g·kg^-1 (i.e., 29.89% increase over control), respectively. Thus, the weightless condition effectively raised the essential AA of F. velutipes 2.82% over control, 8.71% over chicken protein, and 52.63% over the FAO/WHO reference standard. Six protein nutritional indices, including chemical score (CS), AA score (AAS), AA ratio coefficient (AARC), essential AA index (EAAI), biological value (BV) and nutritional index (NI), of the treatment samples were higher than those ofcontrol. Itappeared that the simulated microgravitycultivation increased the AA and protein qualityof F. velutipes fruiting bodies over the conventional method.