The objectives of this study were to determine the effects of in vitro embryo production on histological development and gene expression in the skeletal muscle of bovine fetuses during late gestation. Blastocysts produced in vivo were obtained from superovulated Holstein cows. Blastocysts produced in vitro were obtained from oocytes of Holstein cows that were matured and fertilized in vitro. Single blastocysts were transferred into heifers at a synchronized estrous and fetuses were recovered at Day 222 of gestation (n = 12 each for in vivo and in vitro). Samples of semitendinosus muscle were obtained for histological analysis and assessment of gene expression. Individual muscle sections were stained for the assessment of primary muscle fibers, secondary muscle fibers, or total muscle fibers. Semiquantitative reverse transcription-polymerase chain reaction assays were performed for 5 different candidate genes. The ratio of secondary-to-primary fiber number was greater in fetuses from embryos produced in vitro compared with fetuses from embryos produced in vivo. Similarly, the ratio of secondary-to-primary fiber volume density tended to be greater in fetuses from embryos produced in vitro. The proportional volume of tissue present between myofibrils was greater in fetuses from embryos produced in vitro. The expression of mRNA for myostatin was decreased in skeletal muscle of fetuses in the in vitro group compared with controls. The expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase tended to be increased in skeletal muscle of fetuses in the in vitro treatment group. There was no effect of treatment on the expression of mRNAs for myf-5, myoD, or myogenin. In conclusion, in vitro production of embryos resulted in fetuses with altered development of skeletal muscle fibers. Myostatin was identified as the candidate gene whose expression may contribute to the observed changes in muscle development of these fetuses.