The aim of this study was to evaluate macrophages repolarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype upon transfection with microRNA-223 (miR-223) duplexes and miR-223 expressing plasmid DNA encapsulated in CD44-targeting hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/miR-223 NPs with spherical shape and an average diameter of 200 nm were efficiently internalized by J774A.1 alveolar and primary peritoneal macrophages and non-cytotoxic at HA-PEI concentration less than 200 μg/mL. Transfection of HA-PEI/miR-223 NPs in J774A.1 macrophages showed significantly higher miR-223 expression than that with HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223). HA-PEI/miR-223 NPs mediated transfection increased miR-223 expression to 90 fold in primary peritoneal macrophages compared to untreated cells. The overexpression of miR-223 in both J774A.1 and peritoneal macrophages induced a phenotypic change from M1 to M2 state as indicated by a decrease in iNOS-2 (M1 marker) and an increase in Arg-1 (M2 marker) levels compared to those in lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-stimulated macrophages (M1). The change in macrophage phenotype by HA-PEI/miR-223 NPs could suppress the inflammation in peritoneal macrophages induced by LPS as evidenced by a significant decrease in pro-inflammatory cytokine levels TNF-α, IL-1β and IL-6, compared to LPS-stimulated peritoneal macrophages without treatment. The results demonstrated that miR-223-encapsulated HA-PEI NPs modulated macrophage polarity toward an anti-inflammatory M2 phenotype, which has potential for the treatment of inflammatory diseases.