The biological function of α-lactalbumin (α-LA) depends on its conformation. α-LA can adopt a stable intermediate state induced by heating or pH change. This intermediate state associates with oleic acid (OA) to form an anti-tumor complex. The effect of temperature on the formation the complex has been studied, whereas the effect of pH on complex formation remains unresolved. The effect of pH on tryptophan residues, hydrophobic clusters and secondary structure of Ca(2+)-depleted bovine α-LA (BLA) was studied by fluorescence spectroscopy and circular dichroism. BLA was found to adopt a more flexible conformation between pH 7.0 and 9.0 with buried hydrophobic clusters. The binding ability of α-LA towards OA and the anti-tumor activity of the corresponding complex were also studied. BLA was found to bind more OA over the pH range of 7.0-9.0 and the corresponding complexes showed a higher anti-tumor activity than those complexes formed under acidic conditions. Our study indicates that alkaline pH aided the formation of the complex as well as its anti-tumor activity. We also propose a possible characteristic structure that facilitates binding of OA.