The oxygen isotope composition of diatom frustules is a potential paleoclimate archive; however, the temperature-dependent gradient of the oxygen isotopic records of diatoms is not supported by abiotic experiments. We synthesized amorphous silica under controlled water temperatures (10–40 °C) using the traditional sol-gel method to understand the temperature-dependent oxygen isotopic fractionation. Spherical silica particles were precipitated in the solution containing a relatively low proportion of water, whereas flake-like fragments were precipitated at a high proportion of water (>60 vol. %). The oxygen isotopic values of the flake-shaped silica demonstrated a clear temperature dependence (R2 = 0.87), except for one sample that showed an extremely high value. The observed temperature gradient (–0.092‰/°C) was consistent with the kinetic isotopic fractionation model between orthosilicic acid and water, suggesting two possibilities: 1) spontaneous hydrolysis of tetraethyl orthosilicate to form orthosilicic acid and 2) less temperature dependent isotopic fractionation between orthosilicic acid and amorphous silica. This is the first study to directly evaluate the oxygen isotopic fractionation between synthesized amorphous silica and water, supporting the idea that the temperature-dependent fractionation of oxygen isotopes in freshwater diatoms is based on abiotic processes.