Although radiation-induced bystander effects have been broadly explored in various biological systems, the molecular mechanisms and the consequences of different regulatory factors (dose, time, cell type) on bystander responses are not clearly understood. This study investigates the effects of irradiated cell-conditioned media (ICCM) collected at different times post-irradiation on bystander cancer cells regarding DNA damage and apoptosis induction. Human hepatocellular carcinoma HepG2 cells were exposed to γ-ray doses of 2Gy, 5Gy, and 8Gy. In the early and late stages (1h, 2h, and 24h) after irradiation, the ICCM was collected and transferred to unirradiated cells. Compared to control, bystander cells showed an increased level of H2AX phosphorylation, mitochondrial membrane depolarization, and elevation of intrinsic apoptotic pathway mediators such as p53, Bax, cas9, cas-3, and PARP cleavage. These results were confirmed by phosphatidylserine (PS) externalization and scanning electron microscopic observations, suggesting a rise in bystander HepG2 cell apoptosis. Anti-apoptotic Bcl2-level and viability were lower in bystander cells compared to control. The highest effects were observed in 8Gy γ radiation-induced bystander cells. Even though the bystander effect was persistent at all time points of the study, ICCM at the early time points (1 or 2h) had the most significant impact on the apoptosis markers in bystander cells. Nevertheless, 24h ICCM induced the highest increase in H2AX and p53 phosphorylation and Bax levels. The effects of ICCM of irradiated HepG2 cells were additionally studied in normal liver cells BRL-3A to simulate actual radiotherapy conditions. The outcomes suggest that the expression of the signaling mediators in bystander cells is highly dynamic. A cross-talk between those signaling mediators regulates bystander responses depending on the radiation dose and time of incubation post-irradiation.