d-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient d-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial d-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of d-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24g/L glucose for 9days. The results showed that the production of d-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that d-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a d-mannitol-producing strain.