With the rapid development of dairy industry and improvement of human living standards, the consumption market of dairy products constantly expanding. How to increase the milk yield and optimize milk quality has become the focus of dairy industry. Although numerous microRNA (miRNA) have been verified to play critical roles in the regulation of milk protein synthesis and mammary gland development in cows, but the mechanisms underlying their effects remain unknown. Here, 6 healthy, multiparous Chinese Holstein cows with similar genetic backgrounds were divided into 2 groups according to milk quality (n = 3 per group), statistical analyses were conducted using Student’s test, and P < 0.05 was considered statistically significant. We found that the expression of miR-200b was significantly different between high-milk-quality cows and low-milk-quality ones by using small RNA sequencing and qRT-PCR techniques. Increasing evidences suggested that epigenetic mechanism was involved in the regulation of mammary function in the dairy cow. In recent studies, DNA methylation, as the major epigenetic regulators, has been shown to regulate the expressions of milk proteins in mammary epithelial cells. In vitro, subcultured bovine mammary epithelial cells (BMEC) were cultured in an incubator at 37°C in the presence of 5% CO₂ and were maintained in DMEM/DF-12 with 10% FBS. Our results showed that miR-200b regulated the DNA methylation level of BMECs by inversely targeting both DNMT3A and DNMT3B. We also found that the overexpression of miR-200b promoted BMECs proliferation and significantly decreased the expression of α_S1-casein and β-casein. The treatment of BMECs with 5-aza-2′-deoxycytidine (5-Aza-dC) increased the expression of α_S1-casein and β-casein. This study provided new insight into the molecular mechanisms of miR-200b in regulating lactation performance through DNA methylation, and provided a potential to optimize milk quality not only of the cows but also of their next generations.