Flammulina velutipes is susceptible to mechanical damage, water loss, microbial growth, and other factors that lead to postharvest deterioration, thereby shortening the storage period. The purpose of this study was to analyze the effects of cold plasma treatment on the physicochemical properties and antioxidant capacity of F. velutipes during storage at 4 °C for 21 days. Compared to the control group, cold plasma cold sterilization (CPCS) treatment (150 Hz, 95 kV for 150 s) effectively inhibited the growth and multiplication of microorganisms on the surface of F. velutipes, with no significant effect on the fresh weight change and the superoxide anion generation rate, but with a higher postharvest 1,1-dephenyl-2-picrylhydrzyl (DPPH) clearance rate. Moreover, CPCS increased antioxidant enzyme activities, delayed both malondialdehyde (MDA) accumulation and vitamin C loss, inhibited the browning reaction and polyphenol oxidases (PPO) activity and protected F. velutipes cell membrane from disruption. In general, CPCS not only achieved bacteriostatic effects on F. velutipes during storage, but also reduced cell damage from free radical oxidation, resulting in better postharvest quality and longer shelf life.