Mongolian gerbil 1-cell embryos do not develop into blastocysts in vitro because of 8-cell block. Recently, we reported that 2-cell embryos of Mongolian gerbils developed into blastocysts in vitro in mM16 supplemented with valine. However, the role of valine in the in vitro development of Mongolian gerbil embryos is unclear. Thus, the objective of this study was to investigate of the role of valine in the in vitro development of Mongolian gerbil embryos. Mongolian gerbil embryos at the 1-cell, 2-cell, 8-cell, morula, and blastocyst stages were cultured in mM16 medium containing ¹⁴C-valine or ¹⁴C-glucose. A 2.5 mM NaOH solution was used as a trap for the evolved ¹⁴CO₂. After incubation, all samples were analyzed using a liquid scintillation counter. The incorporation of ¹⁴C-valine significantly increased at the 8-cell stage. The oxidation of ¹⁴C-valine significantly increased at the 8-cell and blastocyst stages. The incorporation of ¹⁴C-glucose in mM16 valine was significantly higher than that in mM16 at the blastocyst stage. The oxidation of ¹⁴C-glucose in mM16 was significantly higher than that in mM16 valine at the 8-cell stage, although that in mM16 valine was significantly higher than that in mM16 at the blastocyst stage. The carbon skeleton of ¹⁴C-valine was metabolized into lipid. Our results suggest that valine was used and metabolized as an energy source in preimplantation Mongolian gerbil embryos.