In order to study the effect of alkali storage on spent mushroom substrate,CaO,Ca (OH)2,CaO+3% urea and Ca(OH)2+3% urea were adopted in the experiment.Each treatment of alkali (CaO/Ca(OH)2) had four levels (3%,4%,5% and 6% additive amount),that was named CO3,CO4,CO5 and CO6;CH3,CH4,CH5 and CH6;COU3,COU4,COU5 and COU6;CHU3,CHU4,CHU5 and CHU6,with 4 replicates per treatment.There were 2 blank groups,one group (CK group) was only spent mushroom substrate,the other one (CKU) was added 3% urea in spent mushroom substrate.Alkaline storage were fermented for 30 d at room temperature.The results showed that compared with the CK group,the content of ADF and free gossypol in CO3,CO4,CO5 and CO6 groups decreased 8.61%,12.69%,13.74%,16.63% and 29.58%,33.92%,39.04%,41.31%;The contents of NDF and ADL in CO5 and CO6 groups were significantly decreased,while the CB2 and RFV were significantly increased (P<0.05).Compared with the CK group,the content of ADF and free gossypol in CH3,CH4,CH5 and CH6 groups were decreased 7.30%,10.90%,13.90%,17.14% and 28.47%,32.69%,40.48%,43.59%,respectively;The contents of NDF and ADL in CH5 and CH6 groups were significant decreased,while the CB2 and RFV were significant increased (P<0.05).Compared with the CKU group,the contents of NDF,ADF and free gossypol in COU3,COU4,COU5 and COU6 groups decreased 6.72%,8.86%, 9.78%,12.10% and 14.63%,16.37%,18.19%,20.21% and 5.49%,14.24%,23.63%,23.66%;RFV increased 18.97%,23.19%,25.86%,30.79%,respectively.Compared with the CKU group,the contents of NDF,ADF,ADL and free gossypol in CHU3,CHU4,CHU5 and CHU6 groups decreased 8.34%,11.87%,14.08%,16.97% and 11.94%,15.19%,18.21%,19.06% and 7.40%,14.93%,19.47%,19.30% and 6.84%,15.60%,22.39%,24.10%,RFV was increased 18.88%,26.20%,34.00%,37.88%, respectively.The effect of CaO and Ca(OH)2 alkali storage was similar.Adding 3% urea alkali to storage much more better than adding CaO or Ca(OH)2,and it could be preserved for a long time.