In a previous genome-wide association study on milk production traits in Chinese Holstein population, we discovered VPS28 gene was highly expressed in mammary gland tissue. Further, a -58C>T mutantion at the 5°-UTR of the gene was significantly associated with milk fat content traits. However, its regulation on milk fat content is still uncertain. In this study, we explored the effect of this -58C>T mutation on VPS28, and found that it could significantly reduce promoter activity of VPS28 gene. To identify the potential function of this SNP, we performed RNAi experiments to knockdown VPS28 gene in BMECs and examined the general effects of VPS28 knockdown on BMECs. The results showed that VPS28 knockdown could decrease the mRNA expression of genes in ubiquitination-lysosome and ubiquitination- proteasome pathways, increase the mRNA expression of genes in milk fat synthesis pathway and promote triglyceride synthesis in BMECs. Our data indicate that VPS28 gene could regulate milk fat synthesis pathway; and promote triglyceride synthesis in BMECs. Our data indicate that VPS28 gene could regulate milk fat synthesis through modulating the ubiquitination-lysosome and ubiquitination-proteasome systems. Our results demonstrate the molecular mechanism of VPS28 on regulation of milk synthesis in dairy cattle through the ubiquitination signaling pathway, thereby supporting a relationship between milk fat synthesis and ubiquitination and laying the molecular foundation in breeding of dairy cattle in the future.