Azithromycin, a macrolide antibacterial, has been shown to modify the phenotype of macrophages. We have investigated whether azithromycin in vitro is able to modulate the differentiation of human blood monocytes to DCs. iA-DCs appear to have a unique phenotype, characterized by increased granularity, adherence, and a surface molecule expression profile similar to that of MDCs, namely, CD1a⁻CD14⁻CD71⁺CD209(high), as well as high CD86 and HLA-DR expression. The iA-DC phenotype is associated with increased IL-6 and IL-10 release, increased CCL2 and CCL18 expression and release, and M-CSF expression, as well as reduced CCL17 expression and release. Upon maturation with LPS, A-DCs and MDCs exhibit decreased expression of HLA-DR and costimulatory molecules, CD40 and CD83, as well as an increase in IL-10 and a decrease in CCL17 and CXCL11 secretion. These modulated responses of iA-DCs were associated with the ability to reduce a MLR, together with enhanced phagocytic and efferocytotic properties. Azithromycin, added 2 h before activation of iDCs with LPS, enhanced IL-10 release and inhibited IL-6, IL-12p40, CXCL10, CXCL11, and CCL22 release. In conclusion, azithromycin modulates the differentiation of blood monocyte-derived DCs to form iA-DCs with a distinct phenotype similar to that of iMDCs, accompanied by enhanced phagocytic and efferocytic capabilities. It also modifies LPS-induced DC maturation by decreasing surface molecule expression required for T cell activation, increasing IL-10 production, and inducing MLR-reducing properties.