Background In vitro fertilization (IVF) is a well-accepted procedure which has been utilized for the treatment of infertile patients. As embryos at early stages of development are very vulnerable, the IVF conditions may influence genetic and epigenetic regulation of preimplantation mouse embryo. Methods We assessed the effect of IVF on the expression of developmental and implantation related miRNAs (miR-21, miR-93, miR-24, and let-7a), their common presumptive target (Stat3), and miRNA biogenesis pathway genes (Drosha, Dgcr8, Exportin-5, Dicer, and Ago2). in vivo 8-cell and blastocysts were compared to IVF embryos. Expression levels of miRNAs, Stat3, and miRNA biogenesis pathway genes were evaluated by qRT-PCR in in vivo (n = 8) and IVF (n = 4) embryos. Results The expression levels of let-7a and Stat3 were significantly reduced in IVF blastocyst when compared with in vivo (p = .004 and p = .009, respectively). Nevertheless, the IVF procedure did not influence the expression levels of miRNA biogenesis pathway components in 8-cell and blastocyst embryos. Conclusions Downregulation of let-7a and developmental related transcription factor, Stat3, in IVF mouse blastocysts may affect preimplantation development and implantation of embryos. Moreover, the genes of the miRNA biogenesis pathway were not changed in preimplantation mouse embryos through the IVF procedure.