E. coli expressed recombinant basic fibroblast growth factor (bFGF) with histidine-tag (bFGF-His) was immobilized onto the surface of a glass plate modified with a Ni(II)-chelated alkanethiol monolayer. The immobilization is expected to take place through the coordination between Ni(II) and His-tag. The bFGF-immobilized surface was exposed to citrate buffer solution to refold in situ the surface-immobilized bFGF. The secondary structure of immobilized bFGF-His was analyzed by solid-phase circular dichroism (CD) spectroscopy. Immortalized human mesenchymal stromal cells (hMSCs) were cultured on the bFGF-His-immobilized surface to examine their proliferation. CD spectroscopy revealed that the immobilized bFGF initially exhibited secondary structure rich in α-helix and that the spectrum was gradually transformed to exhibit the formation of β-strands upon exposure to citrate buffer solution, approaching to the spectrum of native bFGF. The rate of hMSC proliferation was 1.2-fold higher on the bFGF-immobilized surface treated with in situ citrate buffer, compared to the polystyrene surface. The immobilized bFGF-His treated in situ with citrate buffer solution seemed to be biologically active because its secondary structure approached its native state. This was well demonstrated by the cell culture experiments. From these results we conclude that immobilization of bFGF on the culture substrate serves to enhance proliferation of hMSCs.