The objective of this research was to examine the effects of high concentrations of glucose on mouse embryos developing in vitro by studying embryo viability, mitochondrial content and expression of glucose transporters. Addition of 55 mM glucose to the culture medium of two-cell stage embryos significantly reduced the formation of morulae and blastocysts, resulting in fewer cells in the blastocyst stage embryos and increased levels of apoptosis. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression levels of the pro-apoptotic genes Bax and Casp3 at the blastocyst stage were increased significantly by the addition of either 25 or 55 mM glucose to the culture medium. However, addition of 25 or 55 mM glucose to the culture medium did not change the copy numbers of the apoptosis-related miRNAs mmu-mir-15a, mmu-mir-16 and mmu-mir-21. MitoTracker Green fluorescence revealed a decrease in the mitochondrial mass. The expression levels of the mitochondrial DNA-encoded genes Cox1 and Cox2 decreased sharply with the addition of 25 or 55 mM glucose to the culture medium. Both transcripts and protein synthesis of the glucose transporters Glut1 and Glut3 were reduced in blastocysts cultured in the presence of either 25 or 55 mM glucose. These results suggest that hyperglycemia reduces both mitochondrial content and expression levels of glucose transporters in mouse embryos developing in vitro and that this may result in apoptosis in these embryos.