Mongolian gerbil 1-cell embryos can develop into blastocysts in vitro in a co-culture with oviductal cells in mTCM199 medium. When 1-cell embryos are cultured in a chemically defined medium, they do not develop into blastocysts. Thus, the objective of this study was to examine the effects of amino acids on the in vitro development of Mongolian gerbil embryos. Amino acid depletion/accumulation by the Mongolian gerbil preimplantation embryo was analyzed with an amino acid analyzer. The amino acids of threonine, valine, isoleucine, leucine, lysine, serine, glutamine, glycine, and alanine were depleted in the medium at all the developmental stages. Two-cell stage embryos were cultured in mM16 medium supplemented with each amino acid. We found that 37.1% and 11.4% of 2-cell embryos reached the morula and blastocyst stages, respectively, after supplementation of the medium with valine. Three amino acids (threonine, aspartic acid, and glutamine) promoted development to the 4-cell stage (P < 0.05). Although the combination of valine and threonine, aspartic acid, or glutamine significantly increased development to the 4-cell and 8-cell stages, no improvement of the blastocyst rates was observed. This study has demonstrated that amino acids can support preimplantation gerbil embryo development in vitro.