The application of membrane separation of Propionibacterium freudenreichii cells was studied. The effects of membrane material and pore size on the flux and cellular activity were examined. The Vitamin B12 ex-situ transformation was also investigated. The results showed that spiral wound membrane was more suitable for on-line cell separation compared with ceramic membrane. When the fermentation broth was treated with 0.22 mm spiral wound membrane, the biological activity of cells was almost not damaged, with a survival rate of 99.5%, Abo-cbi conversion rate of 78.8% was obtained at 30℃ for 72 h with 4 times concentrated fermentation broth and the productivity of Vitamin B12 reached 53.9 mg/L.