The effects of glutamine supplementation on the blastocoele formation and hatching of mouse preimplantation embryos were investigated. When culturing the 1-cell or 2-cell stage embryos in glutamine-supplemented media, the incidence of development to each stage from the morula stage to the completely hatched blastocyst stage was higher than for culturing in media without glutamine supplementation. When culturing 4-cell stage, 8-cell stage or morula stage embryos in glutamine-supplemented media, percentage of development up to the expanded blastocyst stage was comparable to culturing in media without glutamine supplementation, but the hatching rate for the glutamine-supplemented media was higher or tended to be higher compared to culturing in media without glutamine supplementation. Further, culturing in glutamine-supplemented media from the early blastocyst stage, the blastocyst stage or the expanded blastocyst stage resulted in a higher hatching rate compared to culturing in media without glutamine supplementation. These findings suggest that culturing in glutamine-supplemented media before the 2-cell stage, leads to improvements not only in the rates of morula formation and blastocoele formation, but also the rate of hatching. Further, efficient usage of glutamine appears to be elevated during the course of hatching.