The classical activation of macrophages, one of major innate effector cells, requires IFN-gamma pretreatment (priming) and subsequent TLR stimuli (triggering). The priming effect of IFN-gamma can promote macrophages to secrete higher level of proinflammatory cytokines but lower level of the anti-inflammatory cytokines, enhancing microbicidal and tumoricidal activity of macrophages. However, the underlying molecular mechanisms for IFN-gamma-priming effect on macrophage activation remain to be fully understood. microRNAs (miRNAs) are now emerging as important regulators in immune response, including signaling transduction in immune cell function. In this study, we explored the effect of IFN-gamma on miRNA expression profiling in macrophages and tried to identify the definite miRNA involved in the priming effect of IFN-gamma. We discovered that miR-3473b, which was significantly downregulated after IFN-gamma priming, could attenuate the priming effect of IFN-gamma. miR-3473b promoted Akt/glycogen synthase kinase 3 signaling and IL-10 production through directly targeting phosphatase and tensin homolog (PTEN) to suppress activation of macrophages and inflammatory response. Our data indicate that IFN-gamma beefs up macrophage innate response and cytotoxicity by downregulating miR-3473b to release PTEN from suppression, and then the increase of PTEN contributes to the full activation of IFN-gamma-primed macrophages. Our results provide mechanistic insight to priming effect of IFN-gamma on macrophage classical activation by identifying an IFN-gamma/miR-3473b/PTEN regulatory loop in the regulation of macrophage function.