Background: Bovine mastitis is an inflammatory reaction of the breast caused by pathogenic bacteria infection or breast injury, which affect milk yield and quality and poses severe threat to the health of dairy cows. Pulsatilla chinensis has antibacterial, anti-inflammatory, and immune enhancing effects, etc. However, there was no research about the therapeutic effect of it towards the treatment of cow mastitis. In the study, twelve healthy cows were selected as the control group (group A), and 36 cows with mastitis were equally and randomly divided into group B, C and D. Group A didnât accept treatment, while group B, C and D were treated with 15, 30 and 60 mL Pulsatilla saponin B4 (a purified extract form Pulsatilla chinensis) injection by brachiocephalicus intramuscular injection once a day for 4-6 day and the optimal dose was selected from them. Then, we investigated the effect of this extract on the clearance rates of pathogenic bacteria, the regulation of somatic cell count (SCC) and inflammatory factors the in appropriate dose group.Results: Pulsatilla saponin B4 was effective in all test groups and showed a dose-effect relationship. The extract had inhibitory effects on Streptococcus agalactiae, Streptococcus dysenteriae, Klebsiella species, Mycoplasma bovis, Escherichia coli and Staphylococcus aureus. Group C cured more cows than group B and D during the 6 days of treatment and was determined as the optimal dose group. The milk SCC, serum haptoglobin (HP), prostaglandin E2 (PGE2), interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1ÎČ), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin-10 (IL-10) and tumour necrosis factor alpha (TNF-α), interleukin-4 (IL-4) and leukotriene B4 (LTB4) in Group C were significantly higher than those of healthy cows (P < 0.05) on the first day but had no significant difference with group A after treatment. Conclusion: 30 mL Pulsatilla saponin B4 applied through intramuscular injection once a day for 4-6 days leaded to the significant amelioration of bacterial infection, SCC and levels of serum inflammatory factors in cows with clinical mastitis.