The sphingolipid ceramide is known to play a role in lipid raft fusion and receptor clustering in the plasma membrane (PM). Upon bacterial encounter, dendritic cells (DCs) endocytose the bacteria and initiate a bacteria-specific downstream signaling event. We hypothesized that conversion of sphingomyelin to ceramide by acid sphingomyelinase (ASMase) at the outer leaflet of the PM is a key event in endocytosis of gram-positive Lactobacillus acidophilus (L. acidophilus) and the subsequent induction of IFN-beta in DCs and, as the gram-negative Escherichia coli (E. coli) does not induce appreciable amounts of IFN-beta, the ASMase activity would affect endocytosis and the ensuing cytokine response of L. acidophilus and E. coli differently. SMase or an inhibitor of ASMase and acid ceramidase, chlorpromazine (CPZ), was added to DCs prior to stimulation with either of the bacteria. Endocytosis of fluorescent bacteria +/- FITC-dextran was measured by flow cytometry and gene expression and cytokine response of IFN-beta and IL-12 was measured by qPCR and ELISA, respectively. Addition of SMase increased the uptake of L. acidophilus and L. acidophilus-induced IL-12/IFN-beta but showed no effect on the uptake of E. coli though decreasing IL-12 induced by E. coli. SMase also showed to down-regulate Pam3CSK4-induced macropinocytosis of both bacteria. Addition of CPZ increased actin-dependent uptake of dextran and increased Il-12/Ifn-beta expression induced by L. acidophilus, thus further substantiating the key role of ceramide and thus, phagocytosis, in L. acidophilus-induced IL-12/IFN-beta.