CBLB502 derived from Salmonella flagellin is a novel agonist of Toll-like receptor 5 (TLR5). It has been shown that CBLB502 can exert high radioprotective efficacy on mice and primates from both GI and hematopoietic syndromes during whole-body irradiation with low toxicity and immunogenicity. However, no effective system has been used to investigate the protective effect of CBLB502 against irradiation and the related mechanism in vitro. In this study, we investigated the radioprotective properties of CBLB502 in HEK293-N-T cells constitutively expressing human TLR5 and NF-κB-dependent luciferase. HEK293-N-T cells were treated with different doses of CBLB502 prior to 60Co-γ ray irradiation. After irradiation, cell viability was real-time measured for 4 days by using the real-time cell analysis system. We found that CBLB502 was capable of efficiently maintaining the survival rate of irradiated HEK293-N-T cells. Then apoptotic cell death and cell cycle were detected by flow cytometry. The results showed that CBLB502 pre-treatment could reduce the apoptosis and promote the recovery of irradiated HEK293-N-T cells from G2-phase arrest in a dose-dependent manner. Our data indicated that CBLB502 has a direct radioprotective effect in vitro via anti-apoptosis and promotes cell cycle recovery. The method developed here could be an effective in vitro system to screen other TLR5-target radioprotectants like CBLB502.