Abstract: Objective: To investigate the effects of cell passage number on the establishment of an oxidative stress model in human colon adenocarcinoma cells (Caco-2) caused by tert-butyl hydroperoxide (t-BHP). Methods: First, by applying various t-BHP concentrations (1, 2 and 3 mmol/L) to the F15 generation of Caco-2 cells for 1, 2, 3, 4, 5 and 6 hours, the content of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) in different treatment groups were measured to determine the basic conditions for constructing the oxidative stress model of Caco-2 cells. Then, based on the fundamental parameters, the effect of cell passage number (F14, F15, F18, F19 and F20) on the development of oxidative stress model was investigated using the content of 8-OHdG as an index. Results: When exposed to 1, 2 and 3 mmol/L of t-BHP for 3 to 6 hours, the oxidative stress model of Caco-2 cells from the F15 generation could be established steadily. The content of 8-OHdG in Caco-2 cells of the F14, F15 and F18 generations was significantly higher than that in control group (which had not been treated with t-BHP) after being exposed to 2.0 mmol/L t-BHP for 5 hours (P<0.05), whereas there was no significant difference between the F19 and F20 generations and the control group. Conclusion: The development of the oxidative stress model of Caco-2 cells will be strongly impacted by cell passage number. By subjecting Caco-2 cells prior to F19 to 2 mmol/L t-BHP for 5 hours, the oxidative stress model may be successfully constructed.