The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form. To study adhesion of its FBP (64 kDa), the fbp gene of L. acidophilus NCFM was cloned and expressed in E. coil. However, the fibronectin-binding protein expressed in soluble form could not be purified by Ni-NTA affinity chromatography possibly because of partially buried Histidine tag in the recombinant fusion protein. Therefore, the protein was expressed as inclusion bodies (IBs) at 37 degrees C and solubilized in urea followed by purification in denatured form by Ni-NTA affinity chromatography. The purified denatured protein was refolded in vitro to structurally stable and biologically active form. The conformational properties of the refolded protein were studied by circular dichroism, which showed prominence of alpha + beta structural element. The refolded FBP also showed significant binding to human intestinal tissue sections. Our optimized refolding protocol from IBs of this recombinant probiotic FBP led into high amounts of biologically active protein. Our results help in increasing understanding of structure-function relation of surface adhesion proteins and host-microbial interactions.