Objective:To determine whether the non-adherent mesenchymal stem cells(MSCs) are present in rat peripheral blood and whether 1,25-(OH)2D3 can stimulate them to form colony forming unit-fibroblastic(CFU-f). Methods:Peripheral blood mononuclear and granular cells(PBMGC)fraction was isolated from the blood of six-week old male Wistar rat and were cultured in the absence or presence of 10-8 mol/L 1,25-(OH)2D3 for 16 days After 4 days and 8 days,the non-adherent cells were transferred into another fresh petri dishes to culture consensually until to day 16(“pour-off” culture). The resulting cells were stained with methylene blue and the number of colonies was counted. The resulting cells from 4-day “pour-off” cultures were strained immunocytochemically for vimentin,α-smooth muscle actin(α-SMA),type Ⅰ collagen and type Ⅲ collagen. Results:Not only total PBMGC,but also non-adherent PBMGC can give rise to CFU-fs and the treatment of 10-8 mol/L 1,25-(OH)2D3 increased their CFU-f forming efficacy significantly. The resulting cells from 4-day “pour-off” cultures expressed vimentin,α-SM actin and type Ⅲ collagen,but did not express type Ⅰ collagen. Conclusion:The non-adherent mesenchymal stem cells(MSCs) are present in rat peripheral blood and the treatment of 1,25-(OH)2D3 can stimulate them to form CFU-fs.