Restricting the growth of the embryo can cause adverse whole-of-life changes in an organism's homeostasis. Such adverse long-term consequences may occur even when growth restriction occurs only during the preimplantation period. The molecular basis for these long-term effects has not been defined, although an epigenetic mechanism is suspected. Some loci seem to be more sensitive to epigenetic perturbation than others, and the agouti viable yellow allele (A(vy)) is the best studied example of this. It has active (hypomethylated) and inactive (hypermethylated) epialleles. This study used the A(vy) model to show that growth restriction of preimplantation embryos, as provided by culture of zygotes, induced persistent epigenetic changes that resulted in altered postnatal phenotype. C57BL/6 A(vy)/a males were mated to ovulation-induced FVB/N females, and then either zygotes were collected and cultured for 96 h and the resulting blastocysts were transferred to pseudopregnant recipient females, blastocysts were collected from females and transferred without embryo culture, or pregnancy was allowed to proceed after mating without intervention. Culture was in a commercial human in vitro fertilization media. The proportion of pups expressing the active (hypomethylated) epiallele and yellow coat was significantly higher following zygote culture compared to embryos that were transferred without culture (P = 0.014) or natural matings (P < 0.001). There was no difference in expression of the active epiallele in pups resulting from embryo transfer (without culture) compared to natural matings. These results show for the first time that the preimplantation embryo's growth environment can affect the postnatal expression of a defined epigenetically sensitive allele.