The study was to enhance vitamin K2 production of Flavobacterium sp. by combined mutation of N-methyl-N-nitro-N-nitroso-guanidine (NTG) and low energy N+ ion beam implantation, and optimize the fermentation conditions. The best mutation conditions were as follows: induced dose and time of NTG were 0.8 mg/mL and 20 min; induced energy and dose of N+ implantation were 15 keV and 80 (2.6×1013 cm−2). Vitamin K2 production of the mutant strain reached 6.12 mg/L, which was 159% higher than that of the original. After 6 generations of propagation, the mutant strain with high stability was obtained. Optimal fermentation conditions were obtained when 2% seed culture was inoculated into 250-mL flasks with 30 mL fermentation medium (pH=7.0) and incubated at 37 oC and 120 r/min. Meanwhile, after 72 h of fermentation, Arachis hypogaea in dose of 3 mg/mL was added into the medium as a useful inducer. Vitamin K2 production after optimized-fermentation was 31% higher than that of the mutant strain before optimization.Cited