The aim of the present study was to investigate the influence of collagen changes on the tenderness of yak rumen smooth muscle during storage. Yak rumen smooth muscle was stored at 3 ± 1 °C for 7 d and the Warner–Bratzler shear force (WBSF); total, soluble, and insoluble collagen content (TCC, SCC); collagen solubility (CS); and histological structure were monitored. Label-free mass spectrometry was used to validate the changes in collagen. Collagen changes significantly influenced yak rumen smooth muscle tenderness. A significant positive relationship was found between WBSF and TCC, SCC, as well as CS. The label-free mass spectrometry results validated that the collagen of smooth muscle was degraded during storage. Histological analysis revealed that yak rumen smooth muscle structure was destroyed during storage. Smooth muscle underwent a similar change in tenderness as skeletal muscle, and the weakening of connective tissue was the main reason for smooth muscle tenderness development.