The browning and associated toughening of fruiting body stipes are the main causes of declines in the commercial production of yellow Flammulina filiformis. The dynamic metabolic changes from the top to bottom stipe sections of yellow F. filiformisfruiting bodies were investigated by integrating physiological, transcriptomic, and metabolomic analyses. The results indicated that oxidative stress levels gradually increased accompanying the degree of tissue browning and toughening from the top to bottom sections of F. filiformisstipes. In-depth analysis showed that there were remarkable changes in the expression of genes, and the content of metabolites correlated with the primary and secondary metabolism of F. filiformisstipes. Interestingly, the expression levels of genes participating in chitosan biosynthesis and the degree of deacetylation of chitosan increased from top to bottom in F. filiformisstipes, implying that cell wall glycan remodeling may contribute to concomitant toughening of the browning of F. filiformisstipes.