Objective To investigate the possible mechanism of shikonin in inhibiting synovial cell proliferation and inflammatory response and the effect of combining with low-intensity pulsed ultrasound (LI-PUS) in the treatment of rheumatoid arthritis. Methods Abnormal proliferation of mouse synoviocytes were induced by lipopolysaccharide (LPS) in vitro experiments. CCK-8 and ELISA were performed to detect the proliferation of synoviocytes and the expression levels of inflammatory factors and antioxidants. Cell scratch assay was used to detect the migration of synoviocytes. Immunofluorescence was used to detect the expression of nuclear factor (NF)-κB in synoviocytes. In vivo experiments, a mouse rheumatoid arthritis (RA) model was induced by complete Freund’s adjuvant. Forty mice were equally divided into 4 groups, which were normal group, model control group, shikonin perilla treatment group, and shikonin perilla+LI-PUS combination treatment group. The arthritic swelling indexes of the mice were recorded in day 12 and day 24 of treatment. At the end of the experiment, the serum levels of inflammatory factors were tested, and the expression levels of c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and NF-κB in the synovial tissues of the knee joints in mice were detected by Western blot. Results Shikonin inhibited the expression of inflammatory cytokines in LPS-induced synoviocytes. Shikonin intervention was able to significantly reverse LPS-induced oxidative stress in synoviocytes. The migration of synoviocytes were significantly inhibited under the intervention of different concentrations of shikonin. The level of NF-κB protein expression in synovial cells was reduced after the intervention of shikonin. The results of animal experiments showed that adjuvant-induced arthritis mice had significantly higher joint swelling scores, which could be significantly reduced after the intervention of shikonin. Shikonin+LI-PUS group showed good therapeutic effect on mice. Shikonin inhibited the expression of inflammatory cytokines, in which IL-1β was significantly reduced, and the combination of shikonin+LI-PUS showed highly significant anti-inflammatory effect. Western blot results showed that the phosphorylation content of ERK, JNK and NF-κB in the synovial tissue of mice in the model group was significantly higher than that in the normal group, which was significantly suppressed in the shikonin group and the shikonin+LI-PUS combination treatment group, and the shikonin+LI-PUS combination treatment group was superior to the shikonin group. Conclusions Shikonin can inhibit LPS-induced abnormal proliferation of synoviocytes and exert anti-arthritic activity through the ERK-JNK/NF-κB signaling pathway, and the combination of traditional Chinese medicine and low-intensity pulsed ultrasound is superior to the treatment of traditional Chinese medicine alone.