Embryo transfer uses embryos developed in vivo or in vitro for cattle production, however there is a difference in the quality of the embryos obtained by the two methods. This study addresses the differences in gene expression between blastocysts developed in vitro and in vivo. In vivo blastocysts were flushed from the uteri of super-ovulated cows and blastocysts developed in vitro were derived from in vitro matured and fertilized embryos. The same batch of frozen bull sperm was used for insemination and in vitro fertilization. Blastocysts were then subjected to RNA sequencing. Differentially expressed genes upregulated in in vitro blastocysts were annotated to focal adhesion, extracellular matrix (ECM)-receptor interaction, and PI3K-Akt signaling and the genes that were upregulated in in vivo blastocysts were annotated to oxidation-reduction processes, mitochondrion organization, and mitochondrial translation. Although the total cell number of the two types of blastocysts was similar, the mitochondrial quantity (determined by mitochondrial DNA copy numbers and expression levels of TOMM20), and ATP content in the blastocysts were lower in in vivo blastocysts compared with those developed in vitro. In conclusion, RNAseq revealed differential molecular backgrounds between in vitro and in vivo developed blastocysts and mitochondrial number and function are responsible for these differences.