An association between assisted reproductive technology (ART) and neurobehavioral imprinting disorders has been reported in many studies, and it seems that ART may interfere with imprint reprogramming. However, it has never been explored whether epigenetic errors or imprinting disease susceptibility induced by ART can be inherited transgenerationally. Hence, the aim of this study was to determine the effect of in vitro fertilization and embryo transfer (IVF-ET) on transgenerational inheritance in an inbred mouse model. Mice derived from IVF-ET were outcrossed to wild-type C57BL/6J to obtain their female and male line F2 and F3 generations. Their behavior, morphology, histology, and DNA methylation status at several important differentially methylated regions (DMRs) were analyzed by Morris water maze, hematoxylin and eosin (H&E) staining, and bisulfite genomic sequencing. No significant differences in spatial learning or phenotypic abnormality were found in adults derived from IVF (F1) and female and male line F2 and F3 generations. A borderline trend of hypomethylation was found in H19 DMR CpG island 3 in the female line-derived F3 generation (0.40±0.118, P=0.086). Methylation status in H19/Igf2 DMR island 1, Igf2 DMR, KvDMR, and Snrpn DMR displayed normal patterns. Methylation percentage did not differ significantly from that of adults conceived naturally, and the expression of the genes they regulated was not disturbed. Transgenerational integrity, such as behavior, morphology, histology, and DNA methylation status, was maintained in these generations, which indicates that exposure of female germ cells to hormonal stimulation and gamete manipulation might not affect the individuals and their descendents.