Objectives: The process of human immune response is affected by CD4+CD25+Foxp3+ regulatory T cells (Tregs). Reduced suppressive function or the amount of Tregs has been reported in autoimmune disorders. Previous research suggested that shikonin had anti-inflammatory effect on collagen-induced arthritis (CIA). However, whether Tregs are involved in the remission of CIA by the treatment of shikonin remains unclear. The purpose of this study was to explore the intervention effect of shikonin on Tregs in CIA mice. Methods: CIA mice were divided into 5 groups. Group I, Group II and Group III were given shikonin 2 mg/kg, 3.5 mg/kg and shikonin 5 mg/kg, respectivelly. But Group IV was given meloxicam50 mg/kg, while Group V was CIA diseased mice. Group VI was normal healthy mice. The expression ratio of Tregs and FoxP3 genes was determined by flow cytometry and qRT-PCR. Results: Our results show that the proportion of Tregs was dramatically reduced in CIA diseased mice, while Tregs significantly increased after shikonin treatment. The proportion of Tregs of 5 mg/kg shikonin group was 5.23 times higher than that of CIA diseased group and FoxP3 gene expression was significantly up-regulated 38.82 timesin contrast to the normal healthy group. Conclusions: In brief, shikonin could effectively ameliorate CIA by increasing the proportion of Tregs. The results of this study provide a basis for the change of treatment strategies for human rheumatoid arthritis. © 2020, Fundacion Aigle.