Hypofractionated radiotherapy is a new and highly effective mode of radiation therapy. For this study we used biologically equivalent dose (BED), the dose required to give the same log cell kill as the schedule being studied. BED has been widely accepted to transform its dose to conventionally fractionated ones. However, actual differential effects beyond the clone-forming ability between hypofractionation and conventional radiation treatment remain unknown. We hypothesize that hypofractionation has some advantages over conventional treatment in in vitro radiobiology, excluding influences of the tumor microenvironment in angiogenesis and potential immune-stimulatory effects. For this study, two non-small cell lung cancer (NSCLC) cell lines with different α/β values were chosen: A549 (α/β = 12.4) and H460 (α/β = 2.95). We designed the following two fractionation regimens with equal BED: A549-HRT (10 Gy/1 fraction) and A549-CRT (16 Gy/8 fractions) as well as H460-HRT (10 Gy/1 fraction) and H460-CRT (26 Gy/13 fractions). After irradiation, we performed cell counting, MTT assay, flow cytometry analysis of apoptosis and cell cycle, immunocytofluorescence of γ-H2AX and Hoechst 33258, and senescence-associated β-galactosidase assay to identify differential effects. Glucose consumption and lactic acid production per cell were tested using glucose and lactate assays. Two weeks postirradiation, we collected early-passage cells of the colony cells after both conventional and fractionated irradiations for further investigation. Then, we used the side population (SP) assay, cell-counting assay and Transwell assay to test the proliferation and invasion capability, the MTT assay to identify the drug resistance of cisplatin, pemetrexed and docetaxel, the Western blot assay to test the stem cell-related proteins of NANOG, CD133, OCT4, SOX2, BMI1 and KLF4. After irradiation, the total cell count and cell viability in both cell lines gradually decreased in a similar manner. However, more senescent, necrotic cells and apoptotic cells were found in the conventionally-treated cells at an early time point postirradiation. Contrarily, a higher percentage of G₂/M cell cycle arrest and more γ-H2AX foci were found in the cell lines that received hypofractionated treatment. Glucose consumption and lactic acid production per cell were lower in the cell lines that received hypofractionated irradiation. Early-passage cells in the conventional-treated cell line showed more SP cells with higher expressions of NANOG, OCT4 and BMI1 proteins. Early-passage cells in the conventional-treated cell line also revealed higher proliferative ability, drug resistance and invasion ability. Although we detected some radiobiological differences between the two fractionation treatments, there was no obvious advantage for hypofractionation in the early days postirradiation. However, there were some advantages for hypofractionation compared to conventional treatment in early-passage cells in vitro, which may partially contribute to its clinical advantages. Moreover, the damage to healthy tissue should also be addressed to fully elucidate the implications of radiotherapy addressed in this work.