In this study, we used Raman spectroscopy to observe the process of osteoblast calcification under microvibration stimulation. Osteoblast cultures were prepared under three conditions: the control group was cultured statically without vibration stimulation, and the 45-Hz vibration group and 90-Hz vibration group were stimulated by microvibration at 45 Hz and 90 Hz, respectively, for 30 minutes per day with an acceleration amplitude of 0.2 G. The incubation periods were 7, 9, 11 and 14 days, and observations were conducted using Raman spectroscopy after fixation. In the 45-Hz vibration group, calcification was accelerated. This group produced a large amount of highly crystalline phosphate with large crystal size and a low proportion of hyaluronan precursor and carbonate. In the 90-Hz vibration group, differentiation into mature osteoblasts was not rapid. This group produced more non-crystalline phosphate containing more hyaluronan precursors and carbonate than the other groups. The results suggest that microvibration stimulation promotes calcification, and that the composition of the calcified area may differ depending on the vibration conditions.