In a previous genome-wide association study on milk production traits in Chinese Holstein population, we revealed VPS28 gene was highly expressed in mammary gland tissue and a -58C > T mutation in 5'-UTR of it was significantly associated with milk fat content traits. In this study, we explored the effect of this -58C > T mutation on VPS28, and found it could significantly decrease promoter activity of VPS28 by reducing transcription factor binding sites. To identify the potential functional SNP involved, we performed RNAi experiment in BMECs, the results showed that VPS28 knockdown could increase the expression of ADFP and CD36, lead accumulation of ubiquitinated proteins, long chain fatty acids and triglyceride, and decrease the proteasome activity. Therefore, our study demonstrates that the -58C > T mutation could facilitate milk fat synthesis in two ways. The one is involved in ESCRTs signaling, it could directly lead an accumulation of ubqiuitinated membrane proteins to promote the long chain fatty acids uptake to incorporation into TG. The other is involved in ubiquitination-proteasome system, it could indirectly lead a dysfunction of proteasome to accumulate the ubqiuitinated proteins to promote TG synthesis. In conclusion, our study demonstrates that VPS28 could be a strong candidate gene for milk fat content traits, and in particular, the -58C > T mutation in 5'-UTR of VPS28 could be a functional mutation for its effects on milk fat content. (C) 2019 Published by Elsevier Inc.