Abstract: Proton irradiation with an ultra-high dose rate, known as FLASH, is considered to have a protective effect on normal tissues in clinical practice. To better combine the advantageous technologies of spread-out Bragg peak (SOBP) and ultra-high dose rate irradiation, and to verify whether the proton FLASH effect is effective at the SOBP, 100 MeV protons from the China Institute of Atomic Energy were utilized to conduct SOBP irradiation on human normal liver cells WRL68 and human hepatocellular carcinoma cells Hep G2 at different dose rates. By adjusting the flow intensity of the particle beam output by the accelerator, the conventional dose rate was 0.8 Gy/min, while the FLASH dose rate was 40 Gy/s. At various time points post-irradiation, the CCK8 method was employed to detect cell proliferation, flow cytometry was used to assess cell cycle arrest and apoptosis rate, and the fluorescence method was utilized to measure the content of reactive oxygen species (ROS) in different cells. The experimental results indicate that, at the same irradiation dose at the spread-out Bragg peak, proton FLASH irradiation significantly increases the proliferation activity of normal liver cells and reduces the apoptosis rate. Conversely, for hepatocellular carcinoma cells, proton FLASH irradiation decreases cell proliferation activity and significantly increases the apoptosis rate. Regarding the cell cycle arrest pattern, irradiation at the conventional dose rate is more likely to cause severe G2/M phase arrest, whereas FLASH irradiation exhibites S phase arrest initially, which then shiftes to G2/M phase arrest over time. This shift occurres later in hepatocellular carcinoma cells compared to normal cells. Under FLASH irradiation, the ROS content in both types of cells exhibites an oscillatory trend over time, whereas under the conventional dose rate, it shows a decreasing trend over time. These findings suggest that the protective effect of FLASH on normal tissues remains effective in the region of the SOBP of protons, and it maintains nearly equivalent efficacy in eliminating cancer cells. It also broadens the window of differential responses of the two types of cells over time, which is of great significance in clinical treatment. When combined with the analysis of biological effect indicators, its mechanism of action may be related to the oscillatory changes in the ROS content. The relevant signaling pathways include p53 and others. This study confirms that the proton FLASH irradiation still holds great potential for clinical application in the irradiation mode of the SOBP at the cellular level.