Bovine mastitis is the most common and costly disease of dairy cattle. Cluster of differentiation 4 (CD4) is closely related to the immune response in mastitis. We quantified promoter CpG methylation levels of the CD4 gene in Chinese Holsteins with clinical mastitis (CM) and in healthy controls; these levels were quantitatively detected with bisulfite pyrosequencing assays and confirmed by cloning sequencing. We found that the bovine CD4 promoter had 16% more methyl groups in the cows with CM (75.0 ± 5.8%) compared to the controls (59.0 ± 8.5%). The decreased expression level of CD4 in CM cows may be downregulated by the increased DNA methylation levels in the CD4 promoter. Two-dimensional hierarchical clustering analyses showed large differences in promoter CD4 methylation between mastitic and healthy cows; the dendrogram clearly distinguished the cows with clinical mastitis from healthy controls based on methylation levels.