Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits that are present as the outermost component of the cell wall in several Lactobacillus species. The S-layer proteins have been shown to play a role in the antimicrobial activity of certain lactobacilli. However, it is not fully understood how the S-layer proteins exert this biological function. The aim of this study was to test the hypothesis that Lactobacillus acidophilus S-layer proteins antagonize Salmonella Typhimurium (S. Typhimurium) infection by protecting against F-actin cytoskeleton rearrangements and the activation of mitogen-activated protein kinase (MAPK) signalling pathways. Monolayer transepithelial electrical resistance (TER) was measured after S. Typhimurium infection in Caco-2 cultured human intestinal cells with L. acidophilus S-layer proteins. F-actin rearrangement and MAPK activation were also assessed by immunofluorescence staining or Western blotting. The results showed that when S. Typhimurium was co-incubated with S-layer proteins, the S. Typhimurium-induced Caco-2 cell F-actin rearrangement was reduced, and the S. Typhimurium-induced TER decrease and interleukin 8 (IL-8) secretion were attenuated. Additionally, L. acidophilus S-layer proteins could inhibit S. Typhimurium-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinase (JNK) and p38. This study indicates that L. acidophilus S-layer proteins are able to inhibit S. Typhimurium infection through blocking S. Typhimurium-induced F-actin rearrangements and S. Typhimurium-induced ERK1/2, JNK and p38 activation in Caco-2 cells. These data provide a rationale for the use of lactobacillus S-layer proteins as therapeutic and preventative agents, at least in infectious diarrhoea.