This study was designed to examine a single nucleotide polymorphism (SNP) in the HIF-3α gene in three hundred Changbaishan black cattle using PCR-restriction fragment length polymorphism to determine whether there is an association between this SNP and superovulation. The cloning and sequencing results indicate that the polymorphism is due to a point mutation at the 278-bp position in the HIF-3α gene, resulting in 3 genotypes (AA, AB, and BB). Association analysis indicated that the polymorphism has a significant effect on the number of unfertilized embryos (NUE) (P < 0.05) in the cattle. Cattle with genotype BB had a higher NUE than those with genotype AA, but the difference in NUE between AB and AA or BB was not significant.