We inoculated a spent mushroom substrate from Flammulina velutipes (SMSF) with a microbial additive and assessed the effects on chemical composition, ruminal fermentation parameters, and total-tract nutrient digestibility. In Exp. 1, three cannulated Hanwoo steers were used in an in situ trial to determine the degradation kinetics of dry matter (DM) and crude protein (CP). In Exp. 2, three Hanwoo steers were randomly assigned to experimental diets according to a 3 × 3 Latin square for a 3-week period (2 weeks for adaptation and 1 week for sample collection). Experimental diets included the control diet (3.75 kg/d formulated concentrate mixture + 1.25 kg/d rice straw), SMSF diet (3.19 kg/d formulated concentrate mixture + 1.25 kg/d rice straw + 0.56 kg/d SMSF), and inoculated SMSF (ISMSF) diet (3.19 kg/d formulated concentrate mixture + 1.25 kg/d rice straw + 0.56 kg/d ISMSF). The chemical composition of ISMSF did not differ from that of SMSF. Microbial additive inoculation decreased pH (P<0.05) and improved preservation for SMSF. The percentages of DM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) in ISMSF were slightly lesser than those in SMSF. Ruminal fermentation characteristics and total-tract nutrient digestibility were not affected by diet. Overall, microbial additive inoculation improved preservation for SMSF and may allow improved digestion in the rumen; however, the total digestible nutrients (TDN) of SMSF and ISMSF diets were slightly lesser than the control diet. The ISMSF can be used as an alternative feedstuff to partially replace formulated concentrate feed.