Triuranium octoxide (U 3 O 8 ) is one of the main compounds in the nuclear fuel cycle. As such, identifying its processing parameters that control the oxygen isotopic composition could be developed as a new signature for nuclear forensic investigation. This study investigated the effect of different synthesis conditions such as calcination time, temperature, and cooling rates on the final δ 18 O values of U 3 O 8 , produced from uranium metal, uranyl nitrate hydrate, and uranium trioxide as starting materials. The results showed that δ 18 O of U 3 O 8 is independent of the above-listed starting materials. δ 18 O values of 10 synthetic U 3 O 8 were similar (9.35 ± 0.46‰) and did not change as a function of calcination time or calcination temperature. We showed that the cooling rate of U 3 O 8 at the end of the synthesis process determines the final oxygen isotope composition, yielding a significant isotope effect on the order of 30‰. Experiments with two isotopically spiked 10 M HNO 3 , with a difference of δ 18 O ∼75‰, show that no memory of the starting solution oxygen isotope signature is expressed in the final U 3 O 8 product. We suggest that the interaction with atmospheric oxygen is the main process parameter that controls the δ 18 O value in U 3 O 8 . The uranium mass effect, the tendency of uranium ions to preferentially incorporate 16 O, is expressed during the solid−gas oxygen exchange, which occurs throughout cooling of the system.