Abstract: The aim of the present study was to assess the association between milk production trait and OPN gene polymorphism in Guizhou Holstein Cows. A total of 200 Guizhou Holstein Cows were used as experimental animals; a set of primers were designed to amplify and sequence the OPN gene by PCR-SSCP assay and DNA sequencing technique respectively, and subsequently the polymorphism of the OPN gene were analyzed; the association between milk production trait and OPN gene polymorphism were evaluated by using correlation analysis method. The results showed that there were two alleles for OPN gene including M and N in Guizhou Holstein Cows and their allele frequency was 0.62 and 0.38, respectively; the group polymorphism information content was 0.360, the heterozygosity was 0.689, and M allele was found as the dominant allele in the population; MM type individual had significantly or extremely significantly higher milk fat percentage and 305 days milk yield compared to NN type individual（P〈0.05 or P〈0.01）, and had significantly higher 305 days milk yield compared to MN type individual（P〈0.05）. The combined data suggest that MM genotype of OPN gene may serve as a molecular mark for selection of the traits of high milk fat percentage and milk yield in Guizhou Holstein Cows.