Lactobacillus acidophilus surface layer proteins (SLPs) self-assemble into a monolayer that is non-covalently bound to the outer surface of the cells. There they are in direct contact with the environment, environmental stressors and gut components of the host in which the organism resides. The role of L. acidophilus SLPs is not entirely understood, although SLPs seem to be essential for bacterial growth. We constructed three L. acidophilus L-92 strains, each expressing a mutant of the most abundant SLP, SlpA. Each carried a 12-amino acid c-myc epitope substitution at a different position in the protein. A strain was also obtained that expressed the SlpA paralog SlpB from an originally silent slpB gene. All four strains behaved differently with respect to growth under various stress conditions, such as the presence of salt, ox gall or ethanol, suggesting that SlpA affects stress tolerance in L. acidophilus L-92. Also, the four mutants showed differential in vitro binding ability to human host cell proteins such as uromodulin or dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). Furthermore, co-culture of murine immature DCs with a mutant strain expressing one of the recombinant SlpA proteins changed the concentrations of the cytokines IL-10 and IL-12. Our data suggest that SlpA and SlpB of L. acidophilus participate in bacterial stress tolerance and binding to uromodulin or DC-SIGN, possibly leading to effective immune-modification.