Infrared-visible sum frequency generation vibrational spectroscopy (SFG) was used to characterize the structure of 3-aminopropyltriethoxysilane (APTES) films deposited on solid substrates under controlled experimental conditions for the first time. Our SFG spectra in combination with complementary analytical data showed that APTES films undergo structural changes when cured at an elevated temperature. Before the films are cured, well-ordered hydrophobic ethoxy groups are dominantly present on the surface. A majority of hydrophilic surface amino groups are protonated, and they are either buried or randomly oriented at the interface. After the films are cured, chemically and structurally different neutral amino groups are detected on the surface. Unlike the protonated amino groups, a new class of neutral amino groups is ordered at the interface and shows enhanced reactivity.