High-precision radiotherapy (HPR) has established its important role in the treatment of tumors due to its precise dose distribution. Given its more complicated delivery process, HPR commonly requires more fraction delivery time (FDT). However, it is unknown whether it has an identical response of prolonged FDT on different normal tissues. Our results showed that fractionated irradiation with prolonged FDTs (15, 36, and 50 minutes) enhanced cell surviving fractions for normal tissue cells compared with irradiation with an FDT of 2 minutes. However, the late-responding normal cell line HEI-OC1 was more responsive to prolonged FDTs and demonstrated higher surviving fractions and significantly decreased apoptosis and DNA damage compared to the acute-responding normal cell line HaCaT. Increased autophagy mediated via the ATM-AMPK pathway was observed in HEI-OC1 cells compared with HaCaT cells when irradiated with prolonged FDTs. Furthermore, treatment with the autophagy inhibitor 3-MA or ATM inhibitor KU55933 resulted in enhanced ROS accumulation and attenuation of the effect of prolonged FDT-mediated protection on irradiated HEI-OC1 cells. Our results indicated that late-responding normal tissue cells benefitted more from prolonged FDTs compared with acute-responding tissue cells, which was mainly attributed to enhanced cytoprotective autophagy mediated via the ATM/AMPK signaling pathway.