Myeloid‐derived suppressor cells (MDSC s) and microRNA s (miRNA s) contribute to attenuating immune responses during chronic viral infection; however, the precise mechanisms underlying their suppressive activities remain incompletely understood. We have recently shown marked expansion of MDSC s that promote regulatory T (Treg) cell development in patients with chronic hepatitis C virus (HCV ) infection. Here we further investigated whether the HCV ‐induced expansion of MDSC s and Treg cells is regulated by an miRNA ‐mediated mechanism. The RNA array analysis revealed that six miRNA s were up‐regulated and six miRNA s were down‐regulated significantly in myeloid cells during HCV infection. Real‐time RT ‐PCR confirmed the down‐regulation of miR‐124 in MDSC s from HCV patients. Bioinformatic analysis suggested that miR‐124 may be involved in the regulation of signal transducer and activator of transcription 3 (STAT ‐3), which was overexpressed in MDSC s from HCV patients. Notably, silencing of STAT ‐3 significantly increased the miR‐124 expression, whereas reconstituting miR‐124 decreased the levels of STAT ‐3, as well as interleukin‐10 and transforming growth factor‐β , which were overexpressed in MDCS s, and reduced the frequencies of Foxp3+ Treg cells that were developed during chronic HCV infection. These results suggest that reciprocal regulation of miR‐124 and STAT ‐3 in MDSC s promotes Treg cell development, thus uncovering a novel mechanism for the expansion of MDSC and Treg cells during HCV infection.