Fibroblast growth factor-2 (FGF2) inhibits osteoblast mineralization, but the mechanism by which it does so is not fully understood. Matrix vesicles (MVs) play an essential role in the initiation of mineralization, so the current study examined the effect of FGF2 on the functioning of MVs to investigate this mechanism. This study found that FGF2 significantly inhibited differentiation and mineralization of osteoblast-like Saos-2 cells, as indicated by down-regulation of mRNA expression of the osteogenic master regulator runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and collagen 1 alpha 1 (Colla1), and by decreasing the formation of bone nodules. MVs were isolated from Saos-2 cells cultured in osteogenic medium supplemented with and without FGF2 and their presence was verified using electron microscopy and Western blotting. FGF2 markedly reduced the ALP activity of and in vitro mineralization by MVs. These findings suggest that FGF2 inhibits osteoblast mineralization by limiting the capacity of MVs.