The phosphorylation of EGFR(Thr654) is required for nuclear EGFR importing, and our previous study has shown that pEGFR(Thr654) is an independent prognostic factor for the low survival rate of patients with cervical squamous carcinoma. Now, we aim to examine the role of pEGFR(Thr654) in the activation of DNA-PK and radio resistance. Either CaSki or HeLa cells were exposed to a dose of 4 Gy with a 6 MV X-ray in the presence or absence of Cetuximab or Gefitinib, then EGFR, pEGFR(Thr654), DNA-PKcs and pDNA-PKThr2609 levels were determined using a western blot. DNA damage was quantified with gamma H2AX foci analysis and the response of CaSki and HeLa cells to irradiation was determined using a colony formation assay. In CaSki and HeLa cells, irradiation induced nuclear EGFR accumulation, and pEGFR(Thr654) and pDNA-PKThr2609 levels were both significantly increased. Cetuximab pre-treatment significantly reduced the expression of pEGFR(Thr654) and pDNA-PKThr2609 and enhanced the gamma H2AX foci per cell and sensitivity enhancement ratio in CaSki cells. Gefitinib pre-treatment had a similar but weaker effect. In HeLa cells, similar effects of Cetuximab and Gefitinib on pEGFR(Thr654) and pDNA-PKThr2609 were observed, and no significant difference was found. We found that Cetuximab had a better effect than Gefitinib on attenuating the radio resistance in cervical squamous carcinoma cells via inhibiting pEGFR(Thr654)-mediated nuclear EGFR transport and related DNA-PKT2609-mediated DNA repair. However, in adenocarcinoma cells, both EGFR-targeted drugs had no remarkable effects on the radio sensitivity. Taken together, radiotherapy combined with Cetuximab may be a promising strategy to improve the therapeutic gain for cervical squamous carcinoma patients.