With the in vitro production (IVP) of bovine embryos, a deviation in the expected sex ratio has been reported; the percentage of male embryos is > 50%. The objective of this study was to compare the effects of sperm preparation methods on the sex ratio of IVP bovine embryos. Cumulus oocyte complexes (COCs), recovered from abattoir-derived ovaries, were randomly allocated into two groups after in vitro maturation (IVM) and inseminated with sperm prepared with either a swim-up or Percoll gradient. From Days 1 to 8, presumptive zygotes were cultured in SOFaa medium with 5% fetal calf serum (FCS). Embryos (n = 2044) were sexed by PCR. Only the swim-up method resulted in > 50% male embryos (P < 0.05) on both Days 5 and 8 of culture (59.5 and 58.4%, respectively). Moreover, the swim-up method resulted in a greater percentage of male embryos than Percoll (P < 0.05) on every day analyzed (Day 2, 55.7 vs. 44.1%; Day 3, 53.8 vs. 42.6%; Day 5, 59.5 vs. 45.3%; and Day 8, 58.4 vs. 46.4%). In conclusion, the swim-up method altered the sex ratio of IVP bovine embryos by increasing the percentage of male embryos.