The dry matter and composition of cottonseed hull substrate for P. ostreatus cultivation were tested at different growth stages. As protein, cellulose, hemicellulose and lignin were used by P. ostreatus, the compositional profiles of the substrate were changed greatly, but their rates of change varied at different growing stages. The increase of protein content and the reduction of lignocellulose content contributed to the increase in the dry matter digestibility of the spent substrate, making it possibly acceptable as a potential ruminant feed. This could provide an alternative to environmentally sound use of P. ostreatus spent substrate.