Macrophage polarization plays a crucial role in regulating myocardial inflammation and injuries of coxsackievirus B3 (CVB3)-induced viral myocarditis (VM). It has been reported that miR-223 is a potent regulator of inflammatory responses that involved in macrophage polarization. However, the functional roles of miR-223 in CVB3-induced VM still remain unknown. Here, we found that miR-223 expression was significantly down-regulated in heart tissues and heart-infiltrating macrophages of CVB3-infected mice. Up-regulation of miR-223 in vivo protected the mice against CVB3-induced myocardial injuries characterized by the increased body weight and survival, enhanced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), relieved inflammation, depressed creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and aspartate transaminase (AST) levels, reduced production of interferon (IFN)-γ, interleukin (IL)− 6 as well as increased IL-10. We subsequently found that miR-233 up-regulation significantly suppressed the expression of M1 markers (iNOS, TNF-α and CD 86), and promoted the expression of M2 markers (Arginase-1, Fizz-1 and CD 206) in vivo and in vitro. Furthermore, we confirmed that miR-223 directly targeted Pknox1 to inhibit its expression, and the expression of Pknox1 was inversely correlated with miR-223 expression in heart tissues and heart-infiltrating macrophages of CVB3-infected mice. Gain-of-function analyses indicated that Pknox1 overexpression partially reversed the polarization phenotypes regulated by miR-223 overexpression. Taken together, the data suggest that miR-223 protects against CVB3-induced inflammation and myocardial damage, which may partly attribute to the regulation of macrophage polarization via targeting Pknox1.