Objective To explore the function and mechanism of macrophage subsets in early immune responses to viral infection. Methods Fluorescence-assisted cell sorting was performed to acquire F4 / 80P>hiP> and F4 / 80P>loP> subsets of mouse peritoneal macrophages, which were stimulated with poly(I ∶C) for 8 h. qRT-PCR was performed to compare gene expression levels of pro-inflammatory factors and transcription factors. Furthermore, Western blot was performed to identify the potential activated signaling pathway. Finally, an inhibitor block experiment was conducted. Results The percentage of F4 / 80P>hiP> macrophages was significantly higher than the F4 / 80P>loP> subset (P < 0. 05). After stimulation with poly(I ∶C), gene expression of pro-inflammatory factors interleukin 6 ( IL-6), inducible nitic oxide synthase iNOS, interferon alpha (IFNα), and IFNγ was significantly higher in the F4 / 80P>hiP> subset compared with the F4 / 80P>loP> subset ( P < 0. 05), and expression of transcription factors IRF3 and IRF7 was significantly increased (P < 0. 05). Moreover, phosphorylation of the JNK pathway in the F4 / 80P>hiP> subset was significantly increased (P< 0. 05). Upregulated expression of IL-6 and IFNα were significantly attenuated by SP600125 (P < 0. 05), a phosphorylation inhibitor of the JNK signaling pathway. Conclusions The F4 / 80P>hiP> subset was more activated than the F4 / 80P>loP> subset during the early stage of viral infection, and may be modulated by IRF7 upregulation and JNK pathway activation.