Objective To investigate the feasibility of simulating gravity pressure and observe its effect on preosteoblast OCT1 by centrifugation. Method OCT-1 cells were cultured in 1% agarose gel with a final concentration of 2×107 cells per millilitre. The experiment was divided into two groups according to the duration: one day (1 d) and five days (5 d). Each group was further divided into three subgroups: 0 r/min (control), 200 r/min, and 500 r/min. The 200 r/min group and 500 r/min group were centrifuged for three hours once a day for one day or five days respectively. The control group was kept in the same environment without centrifugation. Results The ColⅠpositive staining was slightly strengthened in the 1d 200 r/min group while it was even more strengthened in the 1d 500 r/min group. Conversely, the positive staining was stronger in the 5d 200 r/min group than that in the 5d 500 r/min group. The markers related to osteoblast differentiation such as Alkaline phosphatise (ALP), Type I collagen α2 (Col1α2), Osteocalcin (OC) and runt-related transcription factor-2 (Runx2) mRNA expression were all upregulated after centrifugation. ConclusionsCentrifugation is a practical method for cell pressure and plays a significant role in promoting the differentiation of preosteoblast OCT-1, which can be used as a new method to simulate the gravity.