Biomaterials with nanostructured surfaces influence cellular response in a significantly different, and often beneficial, manner compared to materials with coarser features. Hydroxyapatite [HA, Ca10(PO4)6(OH)2] and strontium-apatite [Sr10(PO4)6(OH)2] microspheres that present nanotopographies similar to biological apatites were incubated in albumin solutions, at physiological conditions (40 mg ml-1; 37 °C), for up to 72 h. Electronic and vibrational circular dichroism spectroscopies revealed spectral signatures characteristic of stacked β-sheet regions in higher ordered structures (e.g., fibrils). The presence of stacked β-sheets was further evidenced by thioflavin T staining. The sequestration of interfacial Ca atoms by pyrophosphate ions (P2O7 4-), prior to albumin adsorption, prevented stacked β-sheet formation on hydroxyapatite. These results suggest that the charge and/or spatial arrangement of Ca atoms direct stacked β-sheet formation during bovine serum albumin adsorption. Stacked β-sheet spectral features were also observed after incubating HA in fetal bovine serum, highlighting that this phenomena could direct cellular response to these biomaterials in vivo.