Objective To investigate the effect of osteopontin on mouse blastocyst attachment and outgrowth EM>in vitro/EM> and its mechanism. Methods Mouse blastocysts were cultured in microdrops,and the culture plates were precoated with fibronectin. Blastocysts were cultured EM>in vitro/EM> in Quinn’s medium containing various concentrations of recombinant mouse osteopontin, osteopontin antibody and RGD peptide.The percent of hatched blastocysts,blastocysts with adhesion and outgrowth were calculated at 24 hours,48 hours and 72 hours after culture. In another experiment,blastocysts were cultured in 24-well culture plate, the concentrations of matrix metalloproteinases (MMP)-2, -9 of culture medium were detected by ELISA. Results There were no significant difference in percent of hatching and attachment between control and OPN treated groups, however, the rates of outgrowth in OPN treated groups at concentrations of either 1.0mg/L or 10.0mg/L were higher than those in control group after 72 hours culturing(EM>P/EM><0.05 and EM>P/EM><0.01,respectively),and the blastocysts started to outgrowth in advance after 48 hours culturing, whereas mouse osteopontin antibody and RGD reduced the incidence of blastocyst hatching, attachment and outgrowth significantly in a dose-dependent manner. Osteopontin promoted the blastocysts secreting MMP-2, -9 in a dose-dependent and time-dependent manner.Conclusion Osteopontin is involved in regulating embryo early implantation process EM>in vitro/EM> by promoting blastocyst outgrowth and secreting MMP-2, -9.