Mastitis is a common disease occurs in breast-feeding mothers, but published data are poor. This study aimed to study the effects of Tanshinones on treating mastitis. Clinical trials performed in 58 breast-feeding mothers were carried out. B-ultrasound and blood test were used to measure the size of breast mass and the change of blood cell counts. BALB/c mice were injected with LPS and then treated by Tanshinone I or Tanshinone IIA/B. Myeloperoxidase (MPO) activity and the release of inflammatory cytokines were tested by MPO kit, RT-qPCR and ELISA. Mouse mammary epithelial cells (mMECs) were isolated and the effects of Tanshinones were measured by conducting CCK-8 assay, flow cytometry, RT-qPCR and ELISA. Patients treated by Cefprozil combined with Tanshinone got better outcomes than patients treated by Cefprozil alone. In animal trials, Tanshinone I and Tanshinone IIA/B significantly reduced MPO activity, and the levels of TNF-α, IL-1β and IL-6 in serum and mammary gland tissues. In mMECs, Tanshinone I and Tanshinone IIA/B attenuated LPS-induced viability loss and apoptosis. And they effectively inhibited the release of TNF-α, IL-1β and IL-6. Also, Tanshinone I and Tanshinone IIA/B significantly attenuated LPS-evoked NF-κB activation. Tanshinone I and Tanshinone IIA/B have potentials in treating mastitis. The beneficial effects might be through regulating NF-κB activation. Display omitted •Cefprozil plus Tanshinones significantly inhibited mastitis in breast-feeding mothers.•Tanshinone I and Tanshinone IIA/B inhibited LPS-induced mastitis in mice.•Tanshinone I and Tanshinone IIA/B attenuated LPS-induced apoptosis and inflammation in mMECs.•Tanshinone I and Tanshinone IIA/B inhibited NF-κB activation in mMECs.•Tanshinone I and Tanshinone IIA/B have potentials in treating mastitis.