Rice straw was substituted for sawdust at five different ratios of 0, 20%, 40%, 60%, and 80% (Control, RS20, RS40, RS60 and RS80, respectively) to obtain five kinds of Lentinula edodes. The effects of adding cropped rice straw to substrate formulas on the proximate composition and non-volatile taste compounds in mushrooms were investigated. The control group had the highest level of MY and BE among the five formulations. The protein levels in mushrooms decreased with the addition of rice straw and the ash levels increased. We found that trehalose, mannitol, and arabitol were the main soluble sugars in the five kinds of mushrooms. The contents of total free amino acids varied from 16.29 to 24.59 mg/g and the highest level of free amino acids was found in mushrooms cultivated from RS20 and RS40. Moreover, the addition of rice straw improved the contents of monosodium glutamate (MSG)-like amino acids in mushrooms. The 5'-Nucleotide levels ranged from 1.66 to 4.48 mg/g and equivalent umami concentration (EUC) value increased with the addition of rice straw. Our results suggest that rice straw is a potential substitute for sawdust to cultivate L. edodes with more non-volatile taste compounds.