The objective of this study was to examine the effect of the polymorphism in the promoter of toll-like receptor 4 (<i>TLR4</i>) on mastitis and milk production traits. Two novel single nucleotide polymorphisms (SNPs) G-1539A before 5’-upstream region and G+265C in the 5’-UTR of <i>TLR4</i> gene were detected, and genetic association analysis was applied on 421 Chinese Holstein cows. The statistical analysis indicated that two SNPs was significantly associated with 305 Days milk yield (P<0.05) and somatic cell score (SCS) respectively (P<0.01). Our results provide evidence that polymorphisms in the promoter of <i>TLR4</i> are associated with milk production traits and SCS, and may be used as a possible candidate for marker-assisted selection and management in Holstein cattle breeding program.