Our previous studies with clinical data analysis have shown that the proinflammatory factor IL-32 is activated in response to influenza virus infection. However, little is known about how influenza virus induces IL-32 production, and the role of IL-32 in the host immune responses during viral infection remains unclear. In this study, we show that IL-32 production is stimulated by influenza A virus or dsRNA in human PBMCs from healthy volunteers. We demonstrate that the NF-κB and CREB pathways play key roles in the activation of IL-32 production in response to influenza virus infection in A549 human lung epithelial cells. We then show that aberrant epigenetic modifications in the IL32 promoter are important in the transcriptional regulation of IL-32 expression. Interestingly, one CpG demethylation within the CREB binding site increases the binding of CREB to the promoter, which is followed by IL32 transcriptional activation in influenza A virus-infected cells. Overexpression assays combined with RNA interference show that DNA methyltransferases DNMT1 and DNMT3b are critical for IL32 promoter methylation and gene silencing before viral infection. We have demonstrated the anti-influenza virus function of IL-32. Assays for each of the six IL-32 isoforms (α, β, γ, δ, ε, and ζ) during influenza virus infection indicated that all the isoforms have antiviral activity, with different inhibitory rates, and that the effect of IL-32γ is strongest. Our results indicate that the elevated IL-32 levels triggered by influenza virus infection in turn hamper viral replication.