The in vitro experiment was conducted to ensure the supplemental level of spent Flammulina velutipes mushroomsubstrates (SMS) as an energy source in manufacturing of whole crop sorghum silage. Sorghum harvested at heading stage wasensiled with spent mushroom substrates of 20% (S-20), 40% (S-40) and 60% (S-60) as fresh matter basis for 6 week. Theexperiment was conducted by 3, 6, 9, 12, 24, 48 hrs of incubation time with 3 replications. The silages were evaluatedfermentation characteristics and dry matter digestibility (DMD) in vitro. The pH of in vitro solution was inclined to decrease withelapsing the incubation time, and that of the S-20 was significantly (P<0.05) lower than the other treatment at 48 hr ofincubation. Gas production was greater (P<0.05) in the S-20 than the other treatments at 6 and 12 hrs of incubation. Themicrobial growth in vitro was inclined to decrease following 24 hr of incubation, and thereafter sustained the similar levels. Invitro dry matter digestibility (IVDMD) was lowered by increasing the supplemental level of spent mushroom substrate, and was alow level in the S-60 throughout whole incubation time. Althoughthe IVDMD for S-40 was steadily increased from 9 hr ofincubation and reached to similar level with the S-20 at 48 hourof incubation, however SMS for whole crop sorghum silagefermentation might as well add about 20 to 30% in fresh matterbasis when considering DMD.