Alternative substrate materials for mushroom cultivation must be developed due to the unstable supply of the currently used substrates. Threonine acts as a nitrogen source in the substrate used for cultivating mushrooms. Threonine influences food flavor and participates in lipid metabolism and protein synthesis as well as energy metabolism through the tricarboxylic acid cycle. In this study, mushroom cultivation substrate was supplemented with threonine to evaluate the effects of threonine on the growth characteristics of and secondary metabolites produced by Pleurotus ostreatus. Threonine supplementation induced substantial changes in the parameters indicating the quality of fruiting bodies. These findings provide a basis for further research on supplementing substrates with amino acids for cultivating mushrooms.