Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease with the decreasing proportion of regulatory T (Treg) cells. Previous studies have shown that microRNAs (miRNAs, miR) act as key regulators of Treg cells. In this study, we assessed the involvement of miR-143-3p on Treg cells differentiation and function in the RA progress. We reported that the expression of miR-143-3p has been negatively associated with RA disease activity, and actively correlated with anti-inflammatory cytokine IL-10, which was secreted by Treg cells. In vitro, miR-143-3p expression in the CD4+T cells contributed to the upregulation of forkhead box protein 3 (Foxp3), which was the characteristic transcription factor of Treg cells. Notably, miR-143-3p mimics treatment markedly upregulated the frequency of Treg cells in vivo, effectively prevented CIA development and significantly inhibited inflammation in mice. Altogether, we proposed that MiR-143-3p can alleviate CIA by polarizing naive CD4+T cells into Treg cells, which warrants miR-143-3p as a target for the new therapeutic strategy of Treg-deficiency autoimmune diseases such as RA.