Meanquinone-7 (MK-7) can be produced by the fermentation of Bacillus subtilis (natto), which is known for its nutraceutical and pharmacological properties. This study focused on the separation and purification of meanquinone-7 from wet cells produced by Bacillus subtilis (natto) fermentation. Using ethanol to extract menaquinone-7 twice directly from the crude cells after fermentation produced a menaquinone-7 yield of 1.47mg/g. The crude extract was purified using macroporous resin and subsequent crystallization. After treatment with HPD722 resin, the purification of menaquinone-7 were 7.17-fold increased with recovery yields of 97.2%. Menaquinone-7 with purities of more than 96% were produced with recovery yield of 99.3% by recrystallized with 95% ethanol. The developed procedure can produce high-purity of menaquinone-7 as well as high recoveries. This is an economical, efficient and simple process to large-scale preparation of high-purity menaquinone-7.