Type I IFNs (IFN-α/βs) and type III IFNs (IFN-λ1-3) play an important role in host defense against viral infections. The induction of type I IFNs has recently been found to take place also in bacterial infections, and therefore, this study focuses on analyzing the regulation of type III IFNs in response to bacterial stimulation. We found by quantitative RT-PCR that the expression of IFN-λ1 and IFN-λ2/3 mRNAs, as well as that of IFN-β, was similarly up-regulated in response to stimulation with live Salmonella typhimurium or TLR4 agonist LPS in human moDCs. The induction of IFN-λ mRNAs did not require ongoing protein synthesis, and only IFN-λ1 was detected at the protein level. The induction of IFN-λ mRNAs was sensitive to SB202190, Ly294002, and PDTC, which inhibit p38 MAPK, PI3K, and NF-κB activation, respectively. Furthermore, we observed that blocking dynamin-dependent endocytosis pathways with dynasore led to decreased cell surface expression of CD86 and HLA class II molecules and reduced production of IFN-λ1, CXCL10, and IL-6 when the cells were infected with S. typhimurium. Cytokine production was also impaired in dynasore-treated, Streptococcus thermophilus-stimulated cells. Further, inhibition of dynamin prevented S. typhimurium-induced phosphorylation of IRF3 and the internalization of the bacteria. In summary, induction of type III IFNs in bacteria-infected human moDCs requires multiple signaling pathways and involves bacterial phagocytosis.