The nucleation sites of calcium phosphate crystals during collagen mineralization were studied by Fourier transform infrared spectrometry and transmission electron microscopy. It was found for the first time that there is another nucleation site, i.e., carbonyl (>C=O) on collagen, besides the previous reported nucleation site of carboxyl (-COOH). By comparing the IR spectra of collagen not only with collagen/calcium phosphate but also with collagen/Ca2+, it Was observed that the peak intensities of amides I, II, and III of collagen decreased significantly after mineralization. The decrease of the amide I peak intensity was mainly due to blockage of the C=O stretch. Furthermore, the peak for amide I gradually shifted to a lower wavenumber during collagen mineralization. This shift indicated that chemical interaction between carboxyl groups and Ca2+ ions formed in the mineralization.