Recognition of viral nucleic acids leads to type I and type III IFN gene expression and activation of host antiviral responses. At present, type III IFN genes are the least well-characterized IFN types. Here, we demonstrate that the p38 MAPK signaling pathway is involved in regulating IFN-lambda 1 gene expression in response to various types of RNA molecules in human moDCs. Inhibition of p38 MAPK strongly reduced IFN gene expression, and overexpression of p38 alpha MAPK enhanced IFN-lambda 1 gene expression in RNA-stimulated moDCs. The regulation of IFN gene expression by p38 MAPK signaling was independent of protein synthesis and thus, a direct result of RNA stimulation. Moreover, the RIG-I/MDA5-MAVS-IRF3 pathway was required for p38 alpha MAPK to up-regulate IFN-lambda 1 promoter activation, whereas the MyD88-IRF7 pathway was not needed, and the regulation was not involved directly in IRF7-dependent IFN-alpha 1 gene expression. The stimulatory effect of p38 alpha MAPK on IFN-lambda 1 mRNA expression in human moDCs did not take place directly via the activating TBK1/IKK epsilon complex, but rather, it occurred through some other parallel pathways. Furthermore, mutations in ISRE and NF-kappa B binding sites in the promoter region of the IFN-lambda 1 gene led to a significant reduction in p38 alpha MAPK-mediated IFN responses after RNA stimulation. Altogether, our data suggest that the p38 alpha MAPK pathway is linked with RLR signaling pathways and regulates the expression of early IFN genes after RNA stimulation cooperatively with IRF3 and NF-kappa B to induce antiviral responses further.