The objective of the study was to explore the effects of galectin-9 on myeloid suppressor cells in Coxsackievirus B3 (CVB3)-induced myocarditis and the possible mechanisms involved. For this purpose, BALB/c male mice were infected with CVB3 on day 0 and then received intraperitoneal (IP) administration of recombinant galectin-9 or phosphate-buffered saline (PBS) daily from day 3 to day 7. The phenotypes and functions of myeloid suppressor cells were evaluated. The role and mechanism of myeloid suppressor cells and subsets in CVB3-induced myocarditis in vitro were explored. We found that galectin-9 remarkably increased the frequencies of CD11b(+)Gr-1(+) cells in the cardiac tissue and spleen with myocarditis. Ly-6G(+) cells were decreased and Ly-6C(+) cells were increased in galectin-9-treated mice. In addition, CD11b(+)Gr-1(+) cells were highly effective in suppressing CD4(+) T cells. Moreover, our data demonstrate that CD11b(+)Gr-1(+) cells are capable of expanding regulatory T cells (Tregs) from a preexisting population of natural Tregs, which depends on IL-10 but not TGF-. Our results indicate that galectin-9 therapy may represent a useful approach to ameliorate CVB3-induced myocarditis.