A small-silo study was conducted to develop an effective ensiling storage method for the use of cotton waste-based spent Mushroom substrate (SMS) as an animal feed. The SMS was ensiled with 5% molasses (DM basis), 0.5% (v/w) lactic acid bacteria (LAB, Lactobacillus plantarum) inoculant or 0.5% (v/w) yeast (Saccharomyces cerevisiae) inoculant. The treatments included 100% SMS (control), 95% SMS + 5% molasses (T1), 95% SMS + 5% molasses + 0.5% LAB (T2) and 95% SMS + 5% molasses + 5% LAB + 0.5% yeast (T3). The treatments were ensiled for 10. Change in chemical compositions was little (P > 0.05) according to the ensiling process and treatments. Compared with those before ensiling, 100% SMS (control) after ensiling showed unstable fermentative properties with high pH (5.2) and little lactic acid production. Compared with the ensiled control, treatments (T1, T2 and T3) resulted in decreased pH, 18-20 times higher concentrations of lactic acid, and greater populations of total bacteria (P < 0.07), LAB and yeast (P < 0.07). The addition of 5% molasses, 0.5% LAB and 0.5% yeast (T3) to the SMS resulted in the lowest pH (4.25) and the greatest microbial Populations. Treatment T3 was selected for a large scale silo study which was ensiled for 10, 20 and 30 d. As in the small-silo study, the T3 treatment showed favorable fermentative and microbial parameters, compared with the control, by decreasing pH and increasing lactic acid concentrations, LAB and yeast populations. The minimum ensiling period was 20 cl, when pH was reasonably low and LAB and yeast populations were greatest. In conclusion, molasses and microbial inoculation improved silage quality of SMS. (c) 2008 Elsevier Ltd. All rights reserved.