Interferons (IFNs) potentiate macrophage activation typically via a STAT1-dependent pathway. Recent studies suggest a functioning of STAT1-independent pathway in the regulation of gene expression by IFN-γ, thus pointing to the diversity in cellular responses to IFNs. Many functions of IFNs rely on cross-regulation of the responses to exogenous inflammatory mediators such as TLR ligands. Here we investigated the contribution of STAT1-independent pathway to macrophage activation and its underlying mechanism in the context of combined stimulation of IFN and TLR. We found that TLR-induced production of inflammatory cytokines (TNF-α, IL-12) was not simply nullified but was significantly suppressed by signaling common to IFN-γ and IFN-β in STAT1-null macrophages. Such a shift in the suppression of TLR response correlated with a sustained STAT3 activation and attenuation of NF-κB signaling. Using a JAK2/STAT3 pathway inhibitor or STAT3-specific siRNA, blocking STAT3 in that context restored TNF-α production and NF-κB signaling, thus indicating a functional cross-regulation among STAT1, STAT3, and NF-κB. Our results suggest that STAT1 deficiency reprograms IFN signaling from priming toward suppression of TLR response via feedback regulation of STAT3, which may provide a new insight into the host defense response against microbial pathogens in a situation of STAT1 deficiency.