FIP-fve is a low molecular-mass protein identified from Flammulina velutipes, which has many biological activities including immunomodulation. Our previous studies found that FIP-fve had certain growth-promoting effect towards F. velutipes mycelia. In order to explore the native biological function of FIP-fve to F. velutipes, we constructed an over-expression vector pCAMBIA1301-pGPD-FIP-fve, and transformed it into Agrobacterium tumefaciens LBA4404 strain. Finally, FIP-fve gene overexpressed F. velutipe was obtained via Agrobacterium transformation. By comparing the biological characteristics of FIP-fve gene-silenced F. velutipe (RNAi:FIP-fve), FIP-fve gene-overexpressed F. velutipe (pGPD:FIP-fve) and wild F. velutipe (CK) in mycelium and fruiting body stages, we found that the growth speed of pGPD:FIP-fve was faster than that of CK, whereas the growth speed of RNAi:FIP-fve was the lowest. After fruiting body cultivation, related yield traits of pGPD:FIP-fve were significantly better than CK in terms of fruiting body numbers, stalk height, fruiting body yield and biological efficiency, and those traits of RNAi: FIP fve were the worst. In conclusion, these results exhibited that FIP-fve could significantly promote the growth of F. velutipe mycelia and might affect the final yields of F. velutipes. Current paper is the first one to illuminate the native bio-functiont of FIP-fve gene towards F. velutipes, which provides some valuable proofs and references e for comprehensive study and further application of FIPs..