Determining the molecular mechanisms in the regulation of early embryonic development is crucial for assisted reproductive technology clinical applications. Serine/threonine protein kinase 40 (Stk40) is a member of the serine/threonine kinase family. It is essential in diverse signaling pathways associated with a wide range of cellular activities, including proliferation, differentiation, survival and apoptosis. However, its involvement and molecular mechanisms in pre‑implantation embryonic development have not been well‑defined. In the present study, it was demonstrated that Stk40 was involved in the development of mouse pre‑implantation embryos. Immunofluorescence and confocal microscopy analyses showed that Stk40 was equally expressed in the nuclei and cytoplasm during all stages of pre‑implantation mouse embryos of imprinting control region mice. Reverse transcription‑polymerase chain reaction showed a significantly higher transcription rate of Stk40 mRNA in the two‑cell stage. The results demonstrated that Stk40 downregulation by microinjection of small interfering RNA into the mouse zygote markedly decreased the blastulation compared with that in the control (Stk40i‑1 vs. control: 65.2% and 77.0%, P<0.05 and Stk40i‑2 vs. control: 49.8% and 70.1%, respectively, P<0.05). In addition, silencing of Stk40 significantly increased the transcription rate of reticulocalbin‑2, whereas that of the homeobox protein, Cdx2, was decreased. In conclusion, the results suggested that Stk40 may be critical in the development of pre‑implantation embryos.