MYB transcription factors (TFs) play important roles in fungal growth, development, stress response, and secondary metabolism. Cell wall glycan remodeling induced by oxidative damage levels is vital for stipe quality during mature stage of yellow Flammulina filiformis fruiting bodies. In this study, we identified 15 F. filiformis MYB (FfMYB) that are ranging from 28.43 kDa-172.3 kDa, with an average of 73.51 kDa. These FfMYB genes were unevenly distributed among six chromosomes. Phylogenetic analysis indicated that 15 FfMYBs were closely related to existing model fungi, while they were more distant from Arabidopsis thaliana. Based on expression analysis, a MYB TF termed FfMYB13 were isolated and identified as a potential regulator binding the promoter of Ff-FeSOD1, which was negatively correlated with tissue toughening of yellow F. filiformis stipes. The data of DAP-seq analysis suggested that the downstream target genes of FfMYB13 were significantly enriched in cell wall metabolism. The result of EMSA and dual luciferase report experiments demonstrated that FfMYB13 served as an upstream transcriptional regulatory factor that activates four cell wall synthesis metabolism related genes, FfKRE6, Ffgas1, FfHYD-1, and FfGFA1. Moreover, FfMYB13 might negatively influence tissue toughening in the inhibition of oxidative damage by activating Ff-FeSOD1.